Recent advances in targeting the “undruggable” proteins: from drug discovery to clinical trials

pmc Signal Transduct Target Ther Signal Transduct Target Ther 3308 sigtrans Signal Transduction and Targeted Therapy 2095-9907 2059-3635 Nature Publishing Group PMC10480221 PMC10480221.1 10480221 10480221 37669923 10.1038/s41392-023-01589-z 1589 1 Review Article Recent advances in targeting the “undruggable” proteins: from drug discovery to clinical trials Xie Xin 1 2 Yu Tingting 1 Li Xiang 1 Zhang Nan 1 3 Foster Leonard J. 2 Peng Cheng pengcheng@cdutcm.edu.cn 1 http://orcid.org/0000-0003-0832-1864 Huang Wei huangwei@cdutcm.edu.cn 1 http://orcid.org/0000-0002-1536-8882 He Gu hegu@scu.edu.cn 3 1 https://ror.org/00pcrz470 grid.411304.3 0000 0001 0376 205X State Key Laboratory of Southwestern Chinese Medicine Resources, College of Medical Technology and School of Pharmacy, Chengdu University of Traditional Chinese Medicine, 611137 Chengdu, China 2 https://ror.org/03rmrcq20 grid.17091.3e 0000 0001 2288 9830 Michael Smith Laboratories, University of British Columbia, Vancouver, BC V6T 1Z4 Canada 3 grid.412901.f 0000 0004 1770 1022 Department of Dermatology and State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, 610041 Chengdu, China 6 9 2023 2023 8 424931 335 3 4 2023 22 7 2023 2 8 2023 06 09 2023 07 09 2023 05 02 2026 © The Author(s) 2023 https://creativecommons.org/licenses/by/4.0/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ . Undruggable proteins are a class of proteins that are often characterized by large, complex structures or functions that are difficult to interfere with using conventional drug design strategies. Targeting such undruggable targets has been considered also a great opportunity for treatment of human diseases and has attracted substantial efforts in the field of medicine. Therefore, in this review, we focus on the recent development of drug discovery targeting “undruggable” proteins and their application in clinic. To make this review well organized, we discuss the design strategies targeting the undruggable proteins, including covalent regulation, allosteric inhibition, protein–protein/DNA interaction inhibition, targeted proteins regulation, nucleic acid-based approach, immunotherapy and others. Subject terms Medicinal chemistry Target validation https://doi.org/10.13039/501100001809 National Natural Science Foundation of China (National Science Foundation of China) 22177084 82104373 82073998 Zhang Nan Huang Wei He Gu pmc-status-qastatus 0 pmc-status-live yes pmc-status-embargo no pmc-status-released yes pmc-prop-open-access yes pmc-prop-olf no pmc-prop-manuscript no pmc-prop-legally-suppressed no pmc-prop-has-pdf yes pmc-prop-has-supplement no pmc-prop-pdf-only no pmc-prop-suppress-copyright no pmc-prop-is-real-version no pmc-prop-is-scanned-article no pmc-prop-preprint no pmc-prop-in-epmc yes pmc-license-ref CC BY issue-copyright-statement © West China Hospital, Sichuan University 2023 Introduction Owing to the rapid development of molecular biology, tremendous progress has been made in the past decades to uncover key biomacromolecules essential for the occurrence and progression of diseases, providing an effective approach to drug discovery. 1 , 2 These biomacromolecules, including kinases, receptors and channel proteins, are characterized by their tight relation to disease development, specific hydrophobic pockets for binding with ligands, and functional changes after binding. 3 Such targets defined as “druggable,” which means could be targeted pharmacologically, are instrumental to the development of modern medicinal science, leading to evidence-based drug design. 4 , 5 The elucidation of disease mechanisms has been the key to innovative treatments. Thanks to the rise of genomics and proteomics, numerous clinically meaningful targets have been found in human disorders. However, as traditional medicinal chemistry concentrates on druggable targets, increasing disease-related targets have been discovered with few characteristics of conventional druggable targets, namely “undruggable”. 6 – 8 The term “undruggable” refers to target proteins whose functional interfaces are flat and lack defined pockets for ligand interaction, making rational drug design a huge challenge. 6 Despite this, such proteins still belong to drug targets. A typical example of an “undruggable” target is KRAS, one of the most frequently mutated oncogene proteins, with varying mutation rates in different types of solid tumors. It has experienced a long clinical drug vacancy due to its shallow pocket on the surface, which has an undesired polarity. 9 Nevertheless, targeting such undruggable targets has been considered also a great opportunity for treatment of human diseases, and has attracted substantial efforts in the field of medicine. Surprisingly, in 2021, after unremitting efforts, a milestone was achieved: the KRAS G12C inhibitor sotorasib was approved by the FDA for a specific subgroup of patients with non-small cell lung cancer (NSCLC), 10 verifying that targeting “undruggable” proteins is worthwhile. With the deepening research on “undruggable” targets, various molecules sharing similar undruggable features are gradually being divided into the following categories. 3 (1) Small GTPases . The RAS family proteins, including KRAS, HRAS and NRAS, belong to small GTPases. For a long time, these RAS family oncoproteins were considered “undruggable” due to the lack of pharmacologically targetable pockets on surface. Although the stalemate is changing with the emergence of approved preclinical even clinical drugs for specific cancers, drug resistance poses another challenge to the application of KRAS inhibitors. 11 , 12 (2) Phosphatases . As kinases is a classic representative of “druggable” targets with great significance in modulating cell motility, phosphatases are their counterparts, playing a pivotal role in the regulating cellular dynamics by catalyzing the removal of phosphate from proteins, including serine, threonine and tyrosine residues. 13 According to structural characteristics, phosphatases has been classified into two types: protein tyrosine phosphatases (PTPs) and protein serine/threonine phosphatases (PSTPs). Unfortunately, due to the structural similarity sharing within each category of phosphatases, low selectivity and inescapable side effects have greatly hindered the progress of drug discovery. 14 (3) Transcription factors (TFs) . A variety of human disorders are related to dysregulation of TFs involved in numerous biological processes, most of which cannot be targeted by conventional small molecules due to their structural heterogeneity and deficiency of tractable binding sites. 15 , 16 Targeting defined TFs and overcoming drug resistance have been identified as challenging yet promising research hotpots in the medicinal field, particularly in the areas of cancers and neurodegenerative diseases. Notable TFs include p53, Myc, estrogen receptor (ER), androgen receptor (AR), which are involved in the pathological process of neoplasm, X-box-binding protein 1 (XBP1), nuclear factor erythroid 2-related factor (NRF2) in age-related diseases and neurodegenerative diseases, and NF-κB, BTB, CNC homology (BACH), EB, E3 in immunological diseases. Current research is primarily focused on targeting p53 and Myc. 17 (4) Epigenetic targets . Epigenetics refers to heritable changes in gene expression or cellular phenotype that occur without altering the DNA sequence. Epigenetic targets play a crucial role in regulating gene expression patterns and have implications in various biological processes and diseases. The main types of epigenetic modifications include DNA Methylation, Histone Modifications, Non-coding RNAs, Chromatin Remodeling and other Epigenetic Enzymes. Understanding and targeting these epigenetic targets have the potential to unravel the mechanisms underlying various diseases, including cancer, neurological disorders, and cardiovascular diseases. 18 (5) Other proteins . Protein–protein interactions (PPIs) and their networks are of great significance in biological processes and in the regulation of the cell cycle, offering another potential avenue for treatments of complex diseases. RAS and TFs such as p53 and Myc are also subjected to PPI networks. A portion of PPIs, those with flat interaction surfaces, are found to be more difficult to target than other PPIs, making them “undruggable” to a certain extent. Classic PPI-related proteins include anti-apoptotic members of the B-cell lymphoma-2 (Bcl-2) family. Additionally, intrinsically disordered proteins with highly dynamic structures, which interact with various protein partners, are also considered to be undruggable PPI proteins due to a lack of binding cavities. 19 Nowadays, in the face of so-called “undruggable” targets, academia has developed dozens of innovative approaches and pharmaceutical companies have invested billions of dollars, changing the term from “undruggable” to “difficult to drug” or “yet to be drugged,” resulting in several approved drugs and emerging potent chemical entities. 20 – 23 According to the mechanism of undruggable proteins, some major strategies for drug design has been formed correspondingly, including covalent inhibition, allosteric inhibition, PPIs inhibition, targeted proteins regulation, nucleic acid-based approaches, immunotherapy and etc. 3 , 6 By adopting cutting-edge technologies such as fragment-based drug discovery (FBDD), a method leveraging stochastic screening and structure-based design; computer-aided drug design (CADD), simulating and computationally predicting drug-target interactions to screen, design and optimize lead compounds; virtual screening (VS), an in silico screening technique premised on the lock-and-key model of drug–target compatibility; DNA-encoded libraries (DELs), a collection of small molecules conjugated to DNA tags for efficient bio-target screening; targeting allosteric sites, inactivating targets by binding variable loci, etc., strategies for drug design have been well developed systematically. 24 – 26 The form of existing entities includes bifunctional molecules, covalent drugs, peptide-based drugs, protein-based drugs, and therapeutic RNAs. 3 , 6 , 27 In this review, we will illustrate the recent development of drug discovery targeting “undruggable” proteins, according to the types of design strategies. Covalent regulation Covalent inhibitors, also known as irreversible inhibitors, are a class of inhibitors that bind to amino acid residues of target proteins through covalent bonds formed by mildly reactive functional groups to confer additional affinity, compared to that of non-covalent inhibitors, which achieve binding and inhibition of target proteins through non-covalent interactions such as hydrogen bond and van der Waals force, resulting in low selectivity and inhibition ability. 28 – 32 Hence, covalent inhibitors have the advantage of sustained inhibition and a longer residence time compared to non-covalent inhibitors because the covalently bound target is continuously inhibited until protein degradation and regeneration. 28 At the same time, covalent inhibitors can also reduce dosage and improve compliance, avoiding some potential resistance mechanisms. 33 , 34 Due to the recognition of potential benefits of covalency, rational design of covalent drugs has contributed to overcome drug resistance induced by mutated kinases and treat diseases related to hot spot targets. For instance, nirmatrelvir, a part of Paxlovid that has been approved for emergency use in COVID-19, is a covalent inhibitor of M pro of SARS-CoV-2, highlighting the significance of cysteine-reactive covalent functional groups in targeting the protease active site of M pro . 35 As non-covalent interactions are relatively weak, deep grooves on surface of target proteins that allow small molecules to bind effectively are required to guarantee the affinity of non-covalent inhibitors. 36 – 38 Whereas, covalent inhibitors could target undruggable proteins which lack surface “pockets”, offering the potential to expand the therapeutic range. In this area, the approval of the KRAS inhibitor, sotorasib, is a remarkable milestone both in the development of covalent drugs and the progress of drugging the undruggable. Here, we introduce how covalent drugs act on acknowledged undruggable proteins and kinases no longer druggable due to mutations, elaborating on the marketed drugs, drugs in clinical trials and lead compounds developed by the covalent inhibition strategy (Fig. 1 ). Fig. 1 Covalent modulators targeting undruggable proteins. Covalent inhibitors bind to amino acid residues of target proteins through covalent bonds formed by mildly reactive functional groups to confer additional affinity. a Binding modes of selected covalent modulators: covalent KRAS inhibitors bind to the cystine of KRAS G12C mutants to reduces the affinity between GTP and KRAS, thereby locking the KRAS G12C mutant in an inactivated state; covalent EGFR inhibitors bind to Cys797 at ATP binding site of EGFR, showing high affinity for T790M mutants and solving resistance; covalent p53 stabilizers bind to p53-Y220C mutant to restore thermal stability to the wild-type level, or prevent the interaction between MDM2 and p53. b Map of marketed, clinical and preclinical covalent inhibitors in signaling pathways Covalent KRAS inhibitors KRAS plays a crucial role in intracellular signaling pathways that are involved in cell growth and survival. 39 It is the most dominant mutated subtype in the RAS family and is responsible for 85% of RAS gene-driven cancers, particularly in pancreatic, colorectal, and lung cancers. 40 – 42 KRAS alternates between inactive GDP-bound states and active GTP-bound states. KRAS alternates between inactive GDP-bound states and active GTP-bound states, regulated by two types of factors: ① Guanine nucleotide exchange factors (GEFs), such as SOS proteins, which catalyze the transition between KRAS and GTP-bound states; ② GTPase activating proteins (GAPs), which promote the hydrolysis of GTPs bound to KRAS, resulting in the conversion of the active state to one terminating in GDP, thereby inhibiting the activity of KRAS. 43 Targeting KRAS directly presents many difficulties. Its wide range of actions and its normal activity being required for many normal cell functions make it difficult to inhibit. Furthermore, KRAS has high homology with NRAS and HRAS, and its currently known active functional domains of KRAS are mainly pocket-shaped, combining KRAS with either GDP or GTP. 44 Unlike protein kinase, which has a weak affinity with ATP, KRAS has a binding affinity with GTP and GDP at the pM level, making it difficult to compete as effectively as protein kinase inhibitors. In summary, KRAS protein is a featureless, nearly spherical structure with no obvious binding sites, making it difficult to synthesize compounds that can effectively target and inhibit its activity. 45 Long impenetrable, KRAS has become a byword for “undruggable” targets in oncology drug development. Common mutation sites in KRAS include codons 12, 13 and 61, with codon 12 being the most common mutation site. 46 – 48 The most frequent mutant forms were KRAS G12D (41%), KRAS G12V (28%), and KRAS G12C (14%). 49 In recent years, breakthroughs in covalent inhibitors discovered through electrophile-first approaches made it possible to target KRAS G12C mutants. 50 Cysteine is located at codon 12 of KRAS G12C , making it possible to selectively target mutant KRAS covalently. Importantly, KRAS active sites lack cysteine, and KRAS G12C can be specifically inhibited in a covalent manner. In KRAS G12C mutants, small molecules covalently bound to the mutant cystine have been found to bind more readily to GDP-bound KRAS proteins. This binding reduces the affinity between GTP and KRAS, thus preventing GEF from catalyzing the replacement of GDP with GTP, thereby locking the KRAS G12C mutant in an inactivated state. 51 The discovery of this binding “pocket” on KRAS G12C mutants has sparked the development of several small-molecule covalent inhibitors specifically targeting KRAS G12C mutants. Of these, sotorasib and adagrasib are in clinical use, and more than ten are undergoing clinical trials (Table 1 ). Table 1 Covalent modulators targeting undruggable proteins Compound name and structure Target Cancer cell line (activity) Indications Status/clinical trial identifier Ref. Sotorasib (AMG-510) ( 1 ) KRAS G12C – Colorectal cancer, NSCLC Marketed 54 Adagrasib (MRTX-849) ( 2 ) KRAS G12C – NSCLC Marketed 57 JAB-21822 ( 3 ) a KRAS G12C – Colorectal cancer, NSCLC Ongoing NCT05288205 (I/II), NCT05276726 (I/II), NCT05194995 (I/II), NCT05002270 (I/II), NCT05009329 (I/II) 61 JNJ-74699157 (ARS-3248) ( 4 ) a KRAS G12C – Colorectal cancer, NSCLC Completed NCT04006301 (I) 66 Divarasib (RG-6330, GDC-6036) ( 5 ) KRAS G12C – Colorectal tumor, NSCLC Ongoing NCT04449874 (I) 67 D-1553 ( 6 ) a KRAS G12C – Colorectal cancer, NSCLC Ongoing NCT05383898 (I/II), NCT04585035 (I/II), NCT05492045 (I/II), NCT05379946 (I/II) 70 JDQ-443 ( 7 ) KRAS G12C – NSCLC Ongoing NCT05132075 (III), NCT05445843 (II), NCT04699188 (I/II), NCT05329623 (I), NCT05358249 (I/II)) 72 LY-3537982 ( 8 ) a KRAS G12C – Colorectal cancer, NSCLC, etc. Ongoing NCT04956640 (I) 74 BI-1823911 ( 9 ) a KRAS G12C – Biliary cancer, colorectal cancer, NSCLC, etc. Ongoing NCT04973163 (I) 75 BPI-421286 ( 10 ) a KRAS G12C – Advanced solid tumor Ongoing NCT05315180 (I) 77 RMC-6291 ( 11 ) a KRAS G12C – Colorectal cancer, NSCLC, etc. Ongoing NCT05462717 (I) 78 IBI-351 (GFH-925, GF-105) ( 12 ) a KRAS G12C – Colorectal cancer Ongoing NCT05497336 (I), NCT05699993 (I), NCT05688124 (I), NCT05626179 (I), NCT05504278 (I) 79 RM-018 ( 13 ) KRAS G12C H358 (IC 50  = 1.4–3.5 nM) – Preclinical 81 RM-032 ( 14 ) a KRAS G12C – – Preclinical 81 RMC-9805 ( 15 ) a KRAS G12C HPAC (IC 50  = 7 nM) – Preclinical 82 RMC-8839 ( 16 ) a KRAS G12C – – Preclinical 83 6H05 ( 17 ) KRAS G12C – – Preclinical 86 2E07 ( 18 ) KRAS G12C – – Preclinical 86 ARS-853 ( 19 ) KRAS G12C H358 (IC 50  = 1.6 μM) – Preclinical 89 ARS-1620 ( 20 ) KRAS G12C H358 (IC 50  = 0.15 μM) – Preclinical 84 Gray series compounds ( 21 – 23 ) KRAS G12C H358 (IC 50  = 26.6 μM) – Preclinical 90 – 92 G12Si-5 ( 24 ) KRAS G12S A549 (IC 50  = 2.4 μM) – Preclinical 93 G12R inhibitor-4 ( 25 ) KRAS G12R – – Preclinical 94 1_AM, 2_AM, 3_AM, 4_AM ( 26 – 29 ) KRAS G12C H358 (IC 50  = 0.73–2.98 μM) – Preclinical 95 Fell series compounds ( 30 – 33 ) KRAS G12C H358 (IC 50  = 0.07–7.6 μM) – Preclinical 96 Lanman series compounds ( 34 – 36 ) KRAS G12C LNCaP (IC 50  = 0.012–0.211 μM) – Preclinical 98 Shin series compounds ( 37 – 40 ) KRAS G12C MIA PaCa-2 (IC 50  = 0.219–11.4 μM) – Preclinical 97 APG-1842 ( 41 ) a KRAS G12C H358 (IC 50  = 4 nM) – Preclinical 653 EB-160 ( 42 ) a KRAS G12C H358 (IC 50  = 17.54 nM) – Preclinical 654 ERAS-3490 ( 43 ) a KRAS G12C H358 (IC 50  = 1.4–82 nM) – Preclinical 655 VRTX-126 ( 44 ) a KRAS G12C – – Preclinical 656 Afatinib (Giotrit TM , BIBW-2992) ( 45 ) EGFR – Metastatic NSCLC, NSCLC Marketed 122 Dacomitinib (Vizimpro TM , PF-299804) ( 46 ) EGFR – Metastatic NSCLC Marketed 128 Osimertinib (AZD9291) ( 47 ) EGFR – Metastatic NSCLC, NSCLC Marketed 139 Aumolertinib (Almonertinib, HS-10296) ( 48 ) EGFR – Metastatic NSCLC Marketed 141 Lazertinib (YH-25448) ( 49 ) EGFR – Metastatic NSCLC Marketed 142 Alflutinib (Furmonertinib) ( 50 ) EGFR – Metastatic NSCLC Marketed 146 Mobocertinib (TAK-788) ( 51 ) EGFR – Metastatic NSCL Marketed 150 Olmutinib (HM61713, BI-1482694) ( 52 ) EGFR – NSCLC Marketed 152 Neratinib ( 53 ) EGFR – Breast cancer Marketed 155 Pyrotinib (SHR-1258) ( 54 ) EGFR – Breast cancer Marketed 156 Avitinib (Abivertinib, AC0010) ( 55 ) EGFR – Metastatic NSCLC Being applied for approval 159 Oritinib (SH-1028) ( 56 ) EGFR – NSCLC Being applied for approval 163 Sunvozertinib (DZ-0586, DZD-9008) ( 57 ) EGFR – Metastatic NSCLC Being applied for approval 166 Rezivertinib (BPI-7711) ( 58 ) EGFR – Metastatic NSCLC Being applied for approval 169 Olafertinib (CK-101, RX518) ( 59 ) EGFR – NSCLC Completed NCT02926768 (I) 172 Nazartinib (EGF816, NVS-816) ( 60 ) EGFR – Advanced solid tumor, metastatic NSCLC Ongoing NCT03040973 (II) 173 Allitinib (AST-1306) ( 61 ) EGFR – Metastatic breast cancer, NSCLC Ongoing NCT04671303 (II) 177 ES-072 ( 62 ) a EGFR – Metastatic NSCLC Ongoing CTR20180074(I) 182 YK-029A ( 63 ) a EGFR – NSCLC Ongoing CTR20180350(I) 185 Canertinib (CI-1033, PD-183805) ( 64 ) EGFR – Breast cancer, head and neck neoplasms, NSCLC, ovarian cancer Terminated NCT00051051 (II), NCT00174356 (I), NCT00050830 (II) 187 Rociletinib (Xegafri TM , CO-1686) ( 65 ) EGFR – NSCLC Terminated NCT02322281 (III), NCT02186301 (II/III), NCT02147990 (II), NCT02705339 (II), etc. 191 Naquotinib (ASP8273) ( 66 ) EGFR – Metastatic NSCLC, NSCLC Terminated NCT02674555 (I), NCT02588261 (III), NCT03082300 (I), NCT02113813 (II) 195 Mavelertinib (PF-06747775) ( 67 ) EGFR – NSCLC Terminated NCT02349633 (I/II) 198 CL-387785 (EKI-785, WAY-EKI 785) ( 68 ) EGFR A432 (IC 50  = 67 ± 7.6 nM) – Preclinical 199 WZ 4002 ( 69 ) EGFR NIH-3T3 – Preclinical 203 PD series compounds ( 70 – 73 ) EGFR A431 – Preclinical 204 KG13 ( 74 ) p53 Y220C NUGC-4 (IC 50  = 7.1 μM) – Preclinical 214 NPD6878 (Apomorphine) ( 75 ) p53-MDM2 PPI – – Preclinical 218 Hamachi’s research Compound ( 76 ) p53-HDM2 PPI SJSA1, MCF7 – Preclinical 223 MAIM1 ( 77 ) Mcl-1 – – Preclinical 228 PKM2 inhibitor Compound ( 78 ) PKM2 PA-1 (IC 50  = 0.16 μM) – Preclinical 229 Data collected from https://clinicaltrials.gov [last accessed March 2023] a The chemical formula was not disclosed Marketed covalent drugs for KRAS inhibition Sotorasib (AMG-510). In May 2021, the U.S. Food and Drug Administration (FDA) granted accelerated approval to Lumakras (sotorasib, AMG-510), a targeted anticancer drug, for the treatment of NSCLC patients with a KRAS G12C mutation. 52 It also became the first targeted drug for the treatment of KRAS gene mutation in the world, breaking the “undruggable” dilemma and marking a milestone in medical history. In collaboration with Carmot Therapeutics, Amgen investigators have discovered sotorasib (AMG-510) (1), the first selective small molecule KRAS G12C inhibitor to enter clinical trials, through a structure-based design. 53 Sotorasib specifically and irreversibly inhibits KRAS G12C by binding to GDP and locking KRAS in an inactive state. In addition, sotorasib has been shown to strongly inhibit phosphorylation of ERK protein in KRAS G12C cells, thereby suppressing cell proliferation. Current studies on sotorasib have identified a variety of indications for its use in the treatment of adenocarcinoma, metastatic colorectal cancer and metastatic NSCLC. 53 – 55 Subsequently, it has been successively approved for marketing in the European Union, Japan, and other countries. According to the most recent ACCR report, the overall remission rate of sotorasib in patients with KRAS G12C mutated NSCLC was 37%, with a disease control rate of 81%, a median progression-free survival of 6.8 months, and a median duration of remission of 10.0 months. 56 Adagrasib (MRTX-849). At the same time, another high-profile drug targeting the KRAS G12C mutation with impressive clinical data has also stepped up its pace of marketing. Mirati Therapeutics and Array BioPharma have collaborated to identify an irreversible small molecule covalent inhibitor of KRAS G12C , adagrasib (MRTX-849) ( 2 ). 57 Adagrasib binds covalently to Cys12 of KRAS G12C and extends to allosteric pocket S-II P, thereby locking KRAS proteins into inactive conformations and inhibiting RAS/MAPK kinase signaling. 58 At the maximum effective dose of 100 mg kg −1  d −1 , adagrasib demonstrated dose-dependent antitumor effects against different tumor models. Adagrasib is more than 1000 times more selective to KRAS G12C than wild-type KRAS and other proteins containing Cys. It has an oral bioavailability of up to 30%, with a half-life of 25 h after a single dose. 59 At present, the study of adagrasib has revealed its potential for use in the treatment of advanced solid tumors, metastatic colorectal cancer, metastatic NSCLC, and metastatic pancreatic cancer. Adagrasib entered phase III clinical trials in January 2019, and according to the most recent ACCR report, it had an overall remission rate of 58%, with a median duration of treatment of 9.5 months and median duration of remission of 12.6 months. On December 12, 2022, the FDA granted accelerated marketing approval of adagrasib for use in an FDA-approved clinical trial to identify adult patients with locally advanced or metastatic NSCLC with KRAS G12C mutations. 60 Covalent KRAS inhibitors in clinical trials In addition to the marketed drugs, sotorasib and adagrasib, there are currently 10 clinical drugs for covalent RAS inhibitors involving 24 clinical trials, of which 23 are ongoing and 1 has been completed. JAB-21822 ( 3 ) is a small molecule KRAS G12C covalent inhibitor developed by Jacobio. JAB-21822 can lock KRAS G12C in a non-activated state and block the signal transduction of KRAS to the downstream, thus playing an antitumor role. It can be utilized for a multitude of indications, such as colorectal cancer, NSCLC, advanced solid tumor, and metastatic NSCLC, in the clinical research and development stage. 61 , 62 JAB-21822 was enrolled in clinical trials in August 2018. At the 2022 ASCO annual meeting, Jacobio presented phase I clinical data from JAB-21822. As of April 2022, a total of 72 patients with advanced solid tumors had been enrolled in the trial. Among them, 32 patients with KRAS G12C mutation were evaluated for efficacy, with an ORR of 56.3% (18/32) and a disease control rate (DCR) of 90.6% (29/32). In September 2022, the Center for Drug Review (CDE) of the China National Drug Administration approved a pivotal phase II trial of JAB-21822 for second-line and beyond treatment of patients with advanced or metastatic NSCLC with the KRAS G12C mutation. Currently, JAB-21822 is conducting a number of simultaneous Phase I/II clinical trials in China, the United States, and Europe ( NCT05288205 , NCT05276726 , NCT05194995 , NCT05002270 , NCT05009329 ), targeting advanced solid tumor patients with KRAS G12C mutation. Araxes, a subsidiary of Wellspring, was one of the first companies to be involved in the development of new mutation sites for KRAS. JNJ-74699157 ( 4 ), also called ARS-3248, is a new generation, oral, selective, covalent inhibitor of the KRAS G12C subtype developed by this company. It blocks downstream signaling of KRAS G12C by covalently binding to the KRAS G12C complex near S-II P of the KRAS mutant protein. JNJ-74699157 has demonstrated high selectivity for the tumor-associated KRAS G12C protein. 63 – 65 Currently, through clinical research and development, JNJ-74699157 has been found to be effective for advanced solid tumors, metastatic NSCLC, metastatic colorectal cancer, and other indications. In May 2019, Wellspring announced that the FDA had approved an Investigational New Drug (IND) application for JNJ-74699157. Subsequently, JNJ-74699157 conducted a clinical phase I trial ( NCT04006301 ) enrolling patients with KRAS G12C positive advanced solid tumor, which was completed in July 2020 with no results posted. 66 Divarasib ( 5 ) is a small molecule covalent inhibitor of KRAS G12C developed by Genentech that is orally available, highly selective, and potent. It also irreversibly immobilizes KRAS G12C in the inactivation state. Currently, studies on divarasib have found that it can be used in the treatment of NSCLC, advanced solid tumor, colorectal cancer, and other indications. Divarasib was officially enrolled in clinical trials in June 2020, and is currently in phase I clinical trials ( NCT04449874 ) to evaluate its safety, pharmacokinetics and activity in patients with advanced or metastatic solid tumors with KRAS G12C mutations. 67 , 68 Of the 59 patients with NSCLC previously treated with divarasib monotherapy included, 57 patients had evaluable outcomes, 26 of whom were confirmed to be in partial remission (PR), with confirmed objective remission rate (ORR) of 46%. 88.1% of patients experienced at least one adverse event (AE), with the most common AEs being nausea (76.3%), diarrhea (61%), vomiting (54.2%), malaise (23.7%), and loss of appetite (15.3%). Divarasib is more selective than the already marketed sotorasib and adagrasib. According to data presented during the 2022 World Lung Cancer Congress, divarasib treated patients with KRAS G12C mutation NSCLC with ORR of up to 53% (46% of which had been confirmed by imaging). Of the patients tested, 90% had been treated with platinum-based chemotherapy and 86% had received treatment with immune checkpoint inhibitors. 69 D-1553 ( 6 ), an independently developed small molecule KRAS G12C covalent inhibitor by Inventis. Bio., is the first oral antitumor drug targeting KRAS G12C mutation to be approved for clinical trials in China. 70 D-1553 has demonstrated excellent tumor inhibition effect and good safety in preclinical studies, making it an ideal candidate for a variety of clinical indications, such as advanced solid tumors, metastatic colorectal cancer, and metastatic NSCLC. In October 2020, D-1553 was officially registered as ready for clinical trials. Currently, a number of clinical phase I/II trials ( NCT05492045 , NCT05383898 , NCT05379946 , NCT04585035 ) have been initiated to evaluate the application of D-1553 in the combined treatment of NSCLC and in the treatment of solid tumors with IN10018, a highly effective and selective inhibitor of FAK. In 2022, a report on the safety and efficacy of D-1553 was presented at the WCLC Congress. No dose-limiting toxicity of D-1553 was observed in 79 patients with KRASG12C mutant NSCLC. Of these, 3 patients decreased dose due to TRAE, and 2 patients discontinued treatment due to TRAE. Among the 74 patients that could be evaluated, 28 patients had PR, 40 patients had SD, ORR was 37.8% (28/74), and DCR was 91.9% (68/74). 71 JDQ-443 ( 7 ), a selective covalent inhibitor of KRAS G12C developed by Novartis, was officially registered for clinical trials in January 2021. In order to overcome the resistance of other KRAS G12C inhibitors, JDQ-443 covalently binds to the “Switch II pocket” of KRAS G12C and irreversibly locks it into an inactive GDP binding state. Studies on JDQ-443 have revealed that it can be utilized in the treatment of advanced solid tumors, metastatic colorectal cancer, metastatic NSCLC, and other indications. 72 , 73 Furthermore, JDQ-443 in conjunction with the SHP 2 inhibitor TNO-155 has demonstrated a synergistic effect in preclinical animal models, resulting in improved outcomes at lower doses. 73 Preliminary results from a phase I/II trial of JDQ-443 in patients with advanced NSCLC ( NCT04699188 , NCT05132075 , NCT05358249 , NCT05329623 ) indicate an overall response rate of 57% (4/7) in those receiving the recommended dose in the phase II trial. In November 2022, Novartis launched a phase III trial (LBCTR2022055019) to compare the efficacy and safety of JDQ-443 versus TNO-155 in patients with locally advanced or metastatic KRAS G12C mutated NSCLC. Currently, in addition to the suspension of enrollment in the phase I study of JDQ-443 pharmacokinetics in participants with impaired liver function ( NCT05329623 ), other clinical trials are ongoing to evaluate the efficacy of JDQ-443 in patients with locally advanced solid tumors or metastatic KRAS G12C mutations in NSCLC. Unveiled at the 2021 American Association for Cancer Research (AACR) by Lilly, LY-3537982 ( 8 ) is a highly selective and effective covalent KRAS G12C inhibitor. LY-3537982 (IC 50  = 3.35 nM) demonstrated exceptionally high target inhibitory activity in KRAS G12C mutated human H358 lung cancer cell lines, surpassing that of sotorasib (IC 50  = 47.9 nM) and adagrasib (IC 50  = 88.9 nM) by more than 10 and 25 times, respectively. Data from preclinical studies presented at the AACR in 2022 showed that the drug LY-3537982 had good activity, with inhibiting KRAS-GTP binding in lung cancer cell lines carrying the KARS G12C variant. In a variety of mouse tumor models containing KRAS G12C gene variants, LY-3537982 significantly inhibited tumor proliferation or even led to complete tumor regression. Now, LY-3537982 is being developed for the highest stage of research globally for indications including colorectal cancer, NSCLC, ovarian tumors, advanced solid tumors, pancreatic tumors, endometrial cancer, etc. In July 2021, LY-3537982 was registered for clinical trials and is currently in phase I clinical trial ( NCT04956640 ) for KRAS G12C mutant solid tumors. 74 BI-1823911 ( 9 ), developed by Boehringer Ingelheim, is a new molecular entity compound with complete independent intellectual property rights. It is a novel, powerful and highly selective covalent irreversible KRAS G12C oral small molecule inhibitor, intended for the treatment of patients with unresectable, locally advanced, or metastatic solid tumors carrying KRAS G12C specific oncogene mutation. In addition, BI-1823911 is in clinical development for a variety of indications, including adenocarcinoma, metastatic lung cancer, metastatic colorectal cancer, cancer, biliary tract cancer, bile duct cancer, advanced solid tumors, metastatic NSCLC, and metastatic pancreatic cancer. 75 In July 2021, a clinical trial application for BI-1823911 ( NCT04973163 ) began to approve to test different doses of BI-1823911 alone and in combination with other agents in patients with various types of advanced cancer harboring KRAS mutations. 75 The 2022 ACCR Conference focused on the preclinical combination data of BI-1823911 and the SOS1 inhibitor, BI-1701963. When BI-823911 was combined with BI-1701963, an SOS1 inhibitor, a deeper level of PD regulation was observed. By analyzing the dose-and time-dependent combination data of BI-1823911 and KRAS, it was found that BI-1823911 can induce concomitant MAPK pathway regulation, G1 cell cycle arrest, and apoptosis. Furthermore, BI-1823911 demonstrated excellent synergistic anti-proliferation activity when combined with PI3K/mTOR, EGFR inhibitors and SOS1 inhibitors. 76 BPI-421286 ( 10 ) is a newly developed molecular entity by Betta Pharmaceutical with complete independent intellectual property rights. This powerful, highly selective covalent irreversible KRAS G12C oral small molecule inhibitor is intended for the treatment of patients with unresectable, locally advanced, or metastatic solid tumors carrying a KRAS G12C specific oncogene mutation. Preclinical data has demonstrated that BPI-421286 has consistent in vitro and in vivo biological activity, effectively inhibiting the proliferation of tumor cells carrying the KRAS G12C mutation, and exhibiting a good antitumor effect in a variety of transplanted tumor models carrying the KRAS G12C mutation. In April 2022, a phase I clinical trial ( NCT05315180 ) of BPI-421286 was initiated to evaluate its efficacy in an open-marker study in patients with advanced solid tumors. 77 The current inhibitors targeting KRAS G12C are all based on a small molecule-protein binding mechanism that lock KRAS G12C in an inactive state and promotes the depletion of already active KRAS G12C , referred to as the KRAS (OFF) mechanism. However, in the process of GTP conversion to GDP, there are still a small number of active conformations that bind to GTP, giving tumor cells a chance to exploit it. One of the primary reasons why KRAS mutants are difficult to target is that they lack pockets on their surface which would be suitable for binding small molecules, making it difficult to develop effective therapeutic strategies. Research has demonstrated that the activated KRAS protein binds to cyclophilin A, a companion protein, to form pockets that can be targeted by small molecules, providing a potential avenue for the development of a novel type of KRAS inhibitor, aptly named KRAS (ON) inhibitors. The mechanism of KRAS (ON) inhibitors is to prevent cyclophilin A from binding to KRAS in an activated state, thus inhibiting the already activated KRAS from exerting its biological effects and effectively cutting off downstream signaling. This approach may be more effective than KRAS (OFF) inhibitors. 41 Currently, there is a small molecule drug based on RAS (ON) mechanism, RMC-6291 ( 11 ) developed by Warp Drive Bio, which has entered the phase I clinical trial ( NCT05462717 ) in July 2022 for the treatment of solid tumors. RMC-6291 is an orally administered, selective covalent inhibitor designed to treat KRAS G12C -driven mutants in cancer patients. In April 2022, the company reported on the AACR that RMC-6291 demonstrated superior preclinical efficacy compared to adagrasib. 78 IBI-351 (GFH-925, GF-105) ( 12 ), developed by Innovent Biologics, is a novel, irreversible covalent inhibitor of KRAS G12C mutation. It is being developed for indications such as gastrointestinal tumors, NSCLC, solid tumors, solid tumors with KRAS G12C mutations, colorectal cancer, non-squamous NSCLC, etc. In August 2022, IBI-351 was officially registered for clinical trials. Phase I trials of IBI351 in combination with other drugs ( NCT05626179 , NCT05504278 , NCT05497336 , NCT05699993 , and NCT05688124 ) are also underway. For example, the efficacy and safety of IBI-351 in combination with sintilimab ± chemotherapy to treat patients with advanced non-squamous NSCLC of KRAS G12C mutation are being evaluated, as well as the combination of IBI-351and cetuximab in the treatment of KRAS G12C mutated metastatic colorectal cancer. 79 Of the 55 evaluable NSCLC patients, 28 achieved a PR, resulting in an investigator-assessed ORR of 50.9% and DCR of 92.7%. In patients with NSCLC, the ORR assessed by the investigator was 61.9% (13/21) and the DCR was 100% at the recommended dose. 80 Covalent KRAS inhibitors in preclinical research and lead compounds There are also several compounds in preclinical development as covalent inhibitors of various subtypes of RAS (ON). RM-018 ( 13 ), developed by Revolution Medicines, covalently binds to the activated state of KRAS G12C mutants, forms a ternary complex with cyclophilin A and KRAS G12C , thereby inhibiting their activity. Meanwhile, RM-018 retained the ability to bind and inhibit KRAS G12C/Y96D , thus overcoming drug resistance. 81 RM-032 ( 14 ) is another inhibitor of KRAS G12C (ON) mutation, discovered by Jesse Boumelha and his colleagues, with double selectivity for both KRAS G12C (ON) and NRAS G12C (ON). In vitro, RM-032 was shown to improve the persistence of RAS pathway signaling and cell proliferation inhibition in KRAS G12C tumor cells compared to KRAS G12C (OFF) inhibition. RMC-9805 ( 15 ) is a selective, orally-administrated covalent inhibitor of KRAS G12D (ON) inhibitor that has been developed by Revolution Medicines for the treatment of patients with colorectal cancer (CRC), pancreatic cancer, or NSCLC. Studies have demonstrated that RMC-9805 effectively inhibits the growth of KRAS G12D mutant cancer cells, inducing cell apoptosis, with low off-target reactivity. Tumor regression can be achieved by repeated oral administration in a KRAS G12D -driven pancreatic tumor xenograft model. However, it has no inhibitory effect on BRAFV600E dependent cells. 82 RMC-8839 ( 16 ) is the first orally-administered, mutant-selective, covalent KRAS G13C inhibitor developed by Revolution Medicines. This compound directly targets KRAS G13C , an important therapeutic target for patients with lung cancer and some colorectal cancers who are not currently being served by any RAS-targeted drugs. 83 Due to the significance of drug design for RAS, in addition to being inspired by the breakthrough in drugging KRAS, dozens of compounds are in preclinical research, with thousands of compounds being considered as candidates. Of these, ARS-1620 is the first publicly disclosed, drug-like KRAS G12C inhibitor, with profound implications for its development history and significance. 84 In 2012, Kevan M. Shokat, a professor from the University of California, and Troy Wilson, the President and CEO of Kura Oncology, co-founded Araxes Pharma to develop covalent inhibitors targeting the KRAS G12C . In 2013, Shokat and co-workers discovered a new strategy where they used covalent inhibitors to bind to the cysteine of KRAS G12C mutation, and screened out two lead compounds, 6H05 ( 17 ) and 2E07 ( 18 ), by utilizing “tethering” technique. 47 , 85 , 86 In the research of structure-activity relationships of 6H05 derivatives, a new allosteric pocket, S-IIP, was identified in KRAS and exploited in further structural optimization. 59 , 86 Structural analysis showed that 6H05 derivatives formed conformational changes that hindered PPI between RAS and SOS mediated by SW-I and SW-II and further impaired SOS catalyzed nucleotide exchange. PPI between RAS and RAF is also destroyed due to the interruption of residue interaction at the interface and the interruption of the transition between active and inactive forms of RAS. 87 , 88 Moreover, the discovery of allosteric binding site S-II P has become a key point in drug design. The landmark findings are published on Nature. With ongoing development, Wellspring Biosciences—a subsidiary of Araxes Pharma—reported early results with the KRAS G12C inhibitor ARS-853 ( 19 ) in Science and Cancer Discovery in 2016. 89 Due to the undesirable pharmacokinetic properties and poor druggability of ARS-853, they further reported ARS-1620 ( 20 ) on Cell with disclosed structure, which has been embraced as a starting point by numerous drugmakers for further development. The structural optimizations of marketed sotorasib (AMG-510) and adagrasib (MRTX-849), as well as JNJ-74699157 (ARS-3248) in the clinical trial, which are based on a covalent binding strategy, have been inspired by the structure of ARS-1620. To date, dozens of compounds structurally derived from sotorasib (AMG-510), adagrasib (MRTX-849), and ARS-1620 have been developed and patented by various drugmakers, many of which are me-too and fast-follow compounds. Gray et al. developed covalent kinase inhibitors based on GDP/GTP binding sites, providing a new idea for the study of KRAS G12C inhibitors, and obtained a series of nucleotide covalent KRAS G12C inhibitors. 90 They first designed a series of substrate competition-related covalent inhibitors targeting catalytic sites based on their GDP-based structure, and SML-8-73-1 ( 21 ) was identified as the main candidate. In simulated cell conditions, the binding efficiency of SML-8-73-1 was measured in the presence of GDP/GTP of 1 mmol L −1 . The results showed that after incubation for 2 h, the substrate competitive binding of SML-8-73-1 was more than 95% KRAS G12C . However, SML-8-73-1 contains two negatively charged phosphate groups, making it difficult to cross the cell membrane. 86 , 90 Therefore, SM-10-70-1 ( 22 ) was synthesized by modifying phosphoric acid groups of SML-8-73-1 with “caging” technology. 91 SM-10-70-1 showed increased cellular permeability and competitively inhibited KRAS G12C through covalent binding. In addition, KRAS-dependent signaling pathways, such as the Akt and Erk pathways, are also inhibited. Moreover, the ability of SM-10-70-1 to exhibit anti-proliferative activity was demonstrated in several cancer cell lines expressing the KRAS G12C mutation. However, its effective rate and selectivity remain to be further improved. As a result, new SARs research was continued and promising XY-02-075 ( 23 ) was obtained. The chemical and enzymatic stability of XY-02-075 is greatly improved by methylene substitution of the central oxygen in the phosphonic anhydride bonds of SML-8-73-1 and SM-10-70-1. XY-02-075 is expected to be a promising compound despite 40 folds reduction in affinity compared to SML-8-73-1. 92 As KRAS G12C is the most researched mutation subtype, targeting some other mutations of KRAS that do not produce cysteine residues remains a challenge. Fortunately, it was found that nucleophilic residues other than cysteine could be selectively targeted by appropriately introducing covalent warheads. In 2022, Shokat and colleagues reported the development of covalent inhibitors of KRAS G12S mutants and KRAS G12R mutants. 93 , 94 Using adagrasib as the parent core, they introduced a, β-lactone structure that can covalently target serine and successfully developed the first selective covalent inhibitor G12Si-5 ( 24 ) targeting KRAS G12S mutants. G12Si-5 binds to the S-II P domain and inhibits oncogenic signaling, reducing ERK phosphorylation in KRAS G12S mutant cells. The IC 50 value of G12Si-5 in A549 cell line was 2.4 μM. 93 Similarly, they successfully developed KRAS G12R covalent inhibitors, G12R inhibitor-4 ( 25 ), by introducing α, β-diketoamide structures that covalently target arginine. The irreversible reaction of G12R inhibitor-4 combined with mutant arginine residues in S-II P was revealed by X-ray crystal structure, which showed imidazole condensation products formed between the α, β-diketoamide ligand and ε-, η- nitrogen of Arg12. Although arginine residues are less nucleophilic, they can be selectively targeted by small, electronphilic molecular reagents, providing the basis for the development of mutant-specific therapies against KRAS G12R -driven cancers. 94 In addition, various KRAS G12C covalent inhibitors with good clinical application prospects can be further obtained through structural optimization. For example, 1_AM ( 26 ), 2_AM ( 27 ), 3_AM ( 28 ), 4_AM ( 29 ), Fell series compound ( 30–33 ), Lanman series compound ( 34–36 ), Shin series compound ( 37–40 ). 95 – 98 In conclusion, there is still great potential to obtain new covalent KRAS inhibitors through structural optimization. Some representative cases of KRAS G12C covalent inhibitors in preclinical research are listed in (Table 1 ). Covalent EGFR inhibitors Epidermal growth factor receptor (EGFR), a member of the receptor tyrosine kinase family, is a typical transmembrane receptor that initiates signaling cascades upon ligand-stimulated dimerization, thereby activating its tyrosine kinase and multiple downstream effectors. 99 – 101 Moreover, it is involved in embryogenesis and stem cell division, 102 and is implicated in cell proliferation, mitosis, and cancer development. 99 , 103 , 104 Overexpression or increased activity of wild-type EGFR protein can lead to cell proliferation, migration, survival, and anti-apoptosis through signaling cascades, which are strongly associated with the occurrence and development of many cancers, such as NSCLC, breast cancer, glioma, head and neck cancer, cervical cancer, and bladder cancer. 105 – 108 Therefore, EGFR has become a promising target for the design and development of anticancer drugs. Targeted drugs for EGFR are tyrosine kinase inhibitors (TKIs), which inhibit the kinases in the cytoplasm, thus preventing them from activating the EGFR signaling pathway. First-generation EGFR TKIs, such as gefitinib and erlotinib, selectively bind to ATP-binding sites of EGFR tyrosine kinase with non-covalent bond, thereby inhibiting EGFR phosphorylation and significantly delaying disease progression in targeted therapy for NSCLC in the clinic. However, resistance gradually emerged: only 10-19% of patients with advanced non-small cell carcinoma experienced a tumor response to gefitinib; 109 , 110 after using first-generation EGFR TKIs for approximately 9–14 months, almost all tumors progressed again. 111 Afterwards, studies revealed that the reduced sensitivity to gefitinib or erlotinib in NSCLC was linked to EGFR-specific activating mutations. 112 – 116 Mutated EGFR has developed resistance mechanisms to reversible inhibitors, thus limiting drug efficacy and rendering it undruggable. To overcome this problem, irreversible EGFR TKIs, namely second-generation EGFR TKIs, have been designed to covalently bind to the binding site, thus enhancing lasting inhibition of tumor cells. Compared to the first-generation EGFR TKIs, the second-generation EGFR TKIs, such as afatinib, daconmitinib and neratinib, possess an acrylamide Michael receptor side chain that can irreversibly bind to Cys797 at the ATP binding site, showing stronger inhibition effect in clinical practice. However, second-generation EGFR inhibitors still cannot be used to treat patients who develop resistance mutations after first-generation EGFR inhibitors, and can also lead to resistance. 117 These resistances are often associated with T790M mutations, resulting in the development of the third-generation EGFR TKIs, 118 such as WZ 4002, osimertinib, and rociletinib, which were specifically designed for T790M mutants rather than WT-EGFR. including. The third-generation drug retains the acrylamide group and covalently binds to Cys797, but replaces the quinazoline portion of the first- and second-generation compounds with pyrimidine to promote selectivity for T790M, showing a higher affinity for T790M than WT-EGFR. 119 Therefore, developing covalent EGFR inhibitors is highly attractive. Currently, there are several EGFR TKIs on the market, of which second-generation TIKs and third-generation TKIs are covalent inhibitors. Here, we present the development of covalent drugs for EGFR inhibition (Table 1 ). Marketed covalent drugs for EGFR inhibition Afatinib (Giotrit TM , BIBW-2992) ( 45 ) is the first covalent EGFR inhibitor approved by the FDA for lung cancer, and is a second-generation EGFR TKI, which was marketed in July 2013. Similar to the first-generation EGFR TKI, afatinib forms hydrogen bonds to the main chain of Met793 in the hinge region and interacts with hydrophobic regions. The furanyl group is exposed to the solvent, and the 3-chloro-4-fluorophenyl group is located near the “gatekeeper” residue. 50 Clinical trials have demonstrated that afatinib performs better in terms of overall survival than chemotherapy for those with EGFR exon 19 deletion, and provides an overall longer period of effective treatment and good disease control compared to gefitinib, a first-generation EGFR inhibitor. 120 – 123 In addition, afatinib is covalently bound to Cys805 of HER2 and is known as a pan-HER2 inhibitor. However, afatinib showed dose-dependent cytotoxicity by inhibiting WT-EGFR and led to resistance to gefitinib and erlotinib. Furthermore, EGFR exon 20 insertion (ex20ins), which was present in 9.1% of patients with EGFR-mutated NSCLC, was found to be insensitive to afatinib. 124 Dacomitinib (Vizimpro TM , PF-299804) ( 46 ), also an irreversible second-generation EGFR TKI originally developed by Pfizer and co-developed by SFJ Pharmaceuticals in 2012, was approved by the FDA in 2018 for the treatment of metastatic NSCLC with exon 19 deletion and exon 21 replacement. 125 – 128 Dacomitinib has similar binding properties to afatinib, forming hydrogen bonds with hinge residues and hydrophobic interactions with those in the binding pocket. 129 In addition, dacomitinib was found to be more promising for progression-free survival compared to gefitinib in a randomized, phase III clinical trial (ARCHER 1050); however, more severe adverse reactions were observed. 130 As a first-line agent in EGFR mutation-sensitive NSCLC, dacomitinib has been demonstrated by many clinical trials to extend overall survival and show significant advantages over first-generation EGFR TKIs. 127 , 130 As a result, the FDA approved dacomitinib in September 2018 for first-line treatment of advanced NSCLC. Osimertinib (AZD9291) ( 47 ), a third-generation irreversible EGFR TKI currently approved for clinical use, received accelerated FDA approval in November 2015 for second-line treatment of NSCLC, and, subsequently, in 2018, FDA approval for first-line treatment. Based on the pyrimidine ring, osimertinib targets the Cys797 residue at the ATP binding site by forming covalent bonds through unsaturated allyl chains, thus irreversibly binding to the catalytic active center of EGFR kinase and inhibiting the phosphorylation of EGFR and its downstream signaling substrates Akt and Erk. 131 Preliminary clinical studies have shown that osimertinib is capable of inhibiting the L858R mutant of EGFR up to 12 nM, and the IC 50 of L858R/T790M mutant was 1 nM. The inhibition rate of osimertinib against EGFR L858R/T790M mutant was approximately 200-fold higher than that of the wild type. 132 Compared to the standard treatments of erlotinib or gefitin, osimertinib has demonstrated significant benefits in terms of both median progression-free survival and median duration of response in NSCLC patients with EGFR exon deletion 19 or L858R mutations. 133 In addition, multiple studies have demonstrated that osimertinib is able to effectively penetrate the blood-brain barrier, 134 , 135 providing a good therapeutic effect on BMS in advanced NSCLC, and significantly extending progression-free survival in cases of central nervous system (CNS) metastases. 136 , 137 In addition to being used alone, osimertinib is also being studied in combination with other targeted therapies for NSCLC, such as inhibitors of the Met, Bcl-2, and MAPK pathways. 138 – 140 Due to the significant efficacy of osimertinib, contemporaneous to the clinical development of osimertinib, several third-generation EGFR TKIs based on osimertinib structures were also being developed, some of which are also approved. Aumolertinib (Almonertinib, HS-10296) ( 48 ) is an oral, irreversible third-generation EGFR TKI developed by Hansoh Pharmaceuticals. Structurally optimized from osimertinib, aumolertinib introduces a cyclopropyl, which can form hydrophobic interactions with Met790 side chains, to replace the methyl group on the indole ring. This optimization improves inhibitory activity and WT-EGFR selectivity, while simultaneously increasing lipophilicity and blood-brain barrier permeability. 141 It was approved in China in March 2020 and was demonstrated to be a well-tolerated third-generation EGFR TKI, which can be used as a first-line treatment option for EGFR mutated NSCLC, in a phase III trial ( NCT03849768 ) in 2022. However, aumolertinib is still more selective towards mutant EGFR, with semi-inhibitory concentration values for resistant or sensitized EGFR being approximately 2-16 times lower than those for wild-type enzymes. Lazertinib (YH-25448) ( 49 ), an irreversible third-generation EGFR TKI developed by Genosco with strong blood-brain barrier penetration, has been shown to induced dose-dependent regression of subcutaneous and intracranial lesions in mice mutated with EGFR L858R+T790M . It has been shown to have superior efficacy in suppressing tumor growth and improving overall survival compared to the same dose of osimertinib, although adverse reactions were observed. The most common adverse reactions observed were pruritus (12%), decreased appetite (11%), rash (11%), and constipation (10%). The proportion of grade III or higher adverse reactions was 5%. A positive correlation between drug exposure and dose was observed, and no dose-limiting toxicity. 142 – 145 In January 2021, it received marketing approval in South Korea for the treatment of NSCLC. Alflutinib (Furmonertinib) ( 50 ) is a third-generation drug that specifically targets EGFR mutations and was independently developed in China. It is an optimized version of osimertinib, with a few key structural changes. Alflutinib incorporates 2,2,2-trifluoroethyl to replace methyl and introduces an N atom to replace the benzene ring with a pyridine ring. The retained Michael addition acceptor-acrylamide structure allows alflutinib to covalently bind to Cys797 residues, resulting in potent anti-tumor effects. Furthermore, alflutinib’s aminopyrimidine master loop can overcome steric hindrance caused by T790M mutation, while the introduction of the trifluoroethoxy-pyridine structure blocks the production of non-selective metabolites. This enhances alflutinib’s activity and kinase selectivity while reducing off-target effects, leading to fewer side effects. 146 – 148 Alflutinib has high selectivity and strong tumor-shrinking properties, with minimal inhibitory effects on wild-type EGFR. It has been approved in China for treating locally advanced or metastatic NSCLC with EGFR-sensitive mutations since March 2021. In June 2022, it gained first-line indications for EGFR exon 19 deletion (Del19) or exon 21 (L858R) advanced NSCLC, with comparable efficacy to osimertinib. Overall, alflutinib’s unique design and effectiveness make it a promising therapeutic option for EGFR-mutated NSCLC patients. 149 Mobocertinib (TAK-788) ( 51 ) is a novel oral targeted EGFR/HER2 drug, belonging to the fourth-generation of EGFR inhibitors. Structurally similar to osimertinib, it possesses an enhanced inhibitory effect against EGFR exon 20 insertion and other non-sensitive mutations, as well as some inhibitory effect against lung cancer with HER2 exon 20 insertion mutations. 150 , 151 In September 2021, the FDA approved mobocertinib for metastatic NSCLC, advanced NSCLC with EGFR mutation, locally advanced NSCLC, and advanced NSCLC. The drug also received marketing approval in China in January 2023. Olmutinib (HM61713. BI-1482694) ( 52 ) is an orally effective small molecule with potential antitumor activity as a mutation-selective third-generation EGFR inhibitor developed by Hanmi Pharmaceutical Co Ltd for the treatment of NSCLC and lung adenocarcinoma. It binds to cysteine residues near the kinase domain, thereby inducing cell death in tumor cells expressing EGFR. 152 , 153 In May 2016, it was approved for marketing in Korea for the treatment of patients with locally advanced or metastatic NSCLC that is positive for the EGFR T790M mutation. However, as reported by the Korea Ministry of Food and Drug Safety (MFDS) on September 30, 2016, olmutinib resulted in the death of two patients due to severe skin and mucous membrane necrosis during clinical trials, and it has issued a prescribing caution warning against the use of olmutinib in new patients. Following the safety incident, the Korean MFDS issued a statement saying that the adverse event had not been reported in previous clinical trials and that while the clinical use of olmutinib has not been suspended, the Korean approach has recommended that patients who need to use the drug should use it cautiously at the discretion of their doctors. Neratinib ( 53 ) is an oral, potent and irreversible third-generation EGFR TKI that inhibits tumor growth and metastasis by blocking the pan-HER family (HER1, HER2, and HER4) and downstream signaling pathway transduction. This drug is originally developed by Wyeth (now Pfizer) and then Puma Biotechnology. Not only does it competitively occupy the ATP-binding site on EGFR, but it also binds to the unique amino acid residue Cys805 near the opening of the pocket-a homologous cysteine residue to EGFR Cys797-to undergo alkylation or covalent bonding, thus achieving irreversible inhibition of HER2. 154 , 155 Neratinib was approved by the FDA in July 2017 for the treatment of breast cancer, making it the only product in the world approved for intensive adjuvant therapy with trastuzumab (herceptin) in HER2-positive breast cancer to reduce the risk of recurrence. Pyrotinib (SR-1258) ( 54 ), developed by Jiangsu Hengrui Medicine Co Ltd, is an effective, selective and irreversible HER2/EGFR dual-target tyrosine kinase inhibitor with IC 50 values of 38 and 13 nM, respectively. Similarly, as the third-generation EGFR TKI, pyrotinib covalently binds to ATP binding sites in intracellular kinase regions of EGFR, HER2 and HER4, preventing homodimer formation, thereby irreversibly inhibiting autophosphorylation, blocking activation of downstream signaling pathways, and inhibiting tumor cell growth. 156 – 158 It received conditional marketing approval from the National Medical Products Administration (NMPA) in August 2018. Covalent EGFR inhibitors being applied for approval At present, several drugs are in the marketing application stage, such as avitinib, oritinib, sunvozertinib, and rezivertinib. All of these drugs are structurally derived from the third-generation EGFR TKI Osimertinib, which is already available in the market. Avitinib (Abivertinib, AC0010) ( 55 ) is a third-generation, irreversible, mutant-selective EGFR inhibitor. Avitinib forms a covalent bond to C797 in the ATP-binding pocket and has potential antitumor activity. Avitinib inhibits the phosphorylation of EGFR Y1068 and its downstream molecule Akt and extracellular signal-regulated kinase (ERK1/2) in H1975 and HCC827 cells. Moreover, the IC 50 for EGFR L858R/T790M double mutant was 0.18 nM. 159 , 160 Avitinib inhibits cell proliferation, reduces colony formation, and induces apoptosis and cell cycle arrest in ACUTE MYELOGENOUS LEUKEMIA cells, especially those carrying FLT3-ITD mutations. Avitinib is also a novel BTK inhibitor. 161 , 162 Oritinib (SH-1028) ( 56 ) is an irreversible, selective third-generation EGFR TKI. Oritinib overcomes T790M-mediated drug resistance in NSCLC and inhibits WT-EGFR, EGFR L858R , EGFR L861Q , EGFR L858R/T790M , EGFR d746-750 , and EGFR d746-750/T790M kinases. IC 50 were 18, 0.7, 4, 0.1, 1.4 and 0.89 nM, respectively. Oritinib binds irreversibly to EGFR kinase by covalently bonding to form Cys797 residues targeting ATP binding sites. Oritinib effectively and selectively targets mutated EGFR cell lines in vitro. 142 , 163 – 165 Sunvozertinib (DZ-0586, DZD-9008) ( 57 ) is an oral, highly effective and irreversible selective EGFR TKI independently developed by Dizal Pharm Co Ltd. It is the world’s first small molecule compound designed for EGFR/HER2 exon 20 insertion mutation. It has strong activity against a variety of EGFR mutations including EGFR exon 20 insertion mutations and HER2 exon 20 insertion mutations. Sunvozertinib shows strong antitumor activity in cell lines and xenograft models. Additionally, as an oral agent, sunvozertinib demonstrates desirable drug metabolism and pharmacokinetic (DMPK) characteristics in both preclinical and clinical settings. 166 In January 2022, sunvozertinib was granted breakthrough therapy designation by the FDA for the treatment of adult patients with locally advanced or metastatic NSCLC whose disease has progressed during or after prior platinum-containing chemotherapy and who have tested positive for EGFR exon 20 insertion mutations. In September of the same year, Dizal Pharm Co Ltd announced the results of the Chinese registered clinical trial of sunvozertinib in the treatment of EGFR exon 20 insertion (Exon20ins) mutant advanced NSCLC at the European Society of Internal Oncology (ESMO) Congress. The confirmed tumor response rate (ORR) assessed by the Blind Independent Center Evaluation Committee (BICR) was 59.8%, and the registered clinical trial met its primary endpoint. In the follow-up, the company still needs to complete the communication with CDE, submit the new drug marketing application, complete the technical review, on-site verification and other procedures. 167 , 168 Rezivertinib (BPI-7711) ( 58 ) is an orally effective, highly selective and irreversible third-generation EGFR TKI. Rezivertinib shows highly selective inhibitory effects on EGFR Del E746-A750 , EGFR T790M , EGFR L858R/T790M double mutations, including EGFR single mutations, but shows a weak inhibitory effect on WT-EGFR. Rezivertinib has excellent central nervous system (CNS) penetration and antitumor activity. Rezivertinib selectively inhibits the proliferation of EGFR mutated cells in cell lines. 142 , 169 – 171 Covalent EGFR inhibitors in clinical trials Currently, several covalent EGFR inhibitors are undergoing clinical trials, all of which are third-generation or more advanced EGFR TKIs. These inhibitors have demonstrated promising efficacy in inhibiting EGFR mutations. Olafertinib (CK-101/RX518) ( 59 ) is an oral selective EGFR covalent inhibitor approved for second-line treatment in patients with EGFR T790M mutation NSCLC and first-line treatment in patients with EGFR sensitive mutation (Del19, L858R) NSCLC. 172 The drug also shows promise in combination therapy with immune checkpoint inhibitors (PD-1 or PD-L1), c-Met inhibitors, and Mek inhibitors, as demonstrated by preclinical studies. In October 2016, a clinical trial ( NCT02926768 ) began to evaluate the phase I/II study of olafertinib in patients with NSCLC and other advanced solid tumors. In September 2017, the FDA granted Checkpoint Therapeutics orphan drug status for olafertinib in patients with EGFR mutation-positive NSCLC. In 2021, a phase I clinical study (CTR20182402) assessing the safety, tolerability, pharmacokinetics, and initial efficacy of olafertinib in patients with advanced NSCLC was completed, but the results have not been published. In June 2022, the phase I/II study ( NCT02926768 ) was concluded, but the results have not been published yet. A phase III clinical study (CTR20200563) investigating the efficacy and safety of olafertinib in first-line treatment of locally advanced or metastatic NSCLC patients with EGFR mutations is still ongoing. Nazartinib (EGF816, NVS-816) ( 60 ) is a third-generation, covalent, irreversible, and highly selective inhibitor of mutant EGFR, developed by Novartis. 173 This drug specifically targets and inhibits the activity of mutant forms of EGFR, thus preventing EGFR-mediated signal transduction. Nazartinib has been shown to exhibit nanomolar level inhibition of mutant EGFR (L858R, Ex19del) and T790M, demonstrating superior specificity towards mutant EGFR as compared to WT-EGFR. Additionally, it exhibits excellent ADME (Absorption, Distribution, Metabolism, Excretion) and PK (Pharmacokinetics) properties. The drug demonstrates potent inhibitory effects on pEGFR levels in H3255, HCC827, and H1975 cell lines, leading to effective inhibition of cell proliferation. 173 – 176 Although the sponsor withdrew the study of nazartinib and erlotinib/gefitinib in the first-line treatment of locally advanced/metastatic NSCLC with EGFR mutations ( NCT03529084 ) in 2019. The latest study shows that nazartinib continues to be studied as a combination drug in a clinical trial ( NCT03040973 ), which called “Study to allow patients previously participating in a Novartis sponsored trial to continue receiving capmatinib treatment as single agent or in combination with other treatments or the combination therapy alone”. Allitinib (AST-1306) ( 61 ) is an orally available anilino-quinazoline compound with demonstrated anticancer activity. It irreversibly inhibits EGFR with an IC 50 value of 0.5 nM, and also inhibits ErbB2 and ErbB4 with IC 50 values of 3 and 0.8 nM, respectively. In HIH3T3-EGFR T790M/L858R cells, allitinib significantly and dose-dependently inhibited cell growth (0.19–6.25 μM; 72 h). It also inhibited the activation of tyrosine kinase and downstream signaling pathways in A549 cells, Calu-3 cells, and SK-OV-3 cells. In A549 cells, allitinib (0.001–1.0 μM; 4 h) showed 3000-fold selectivity to ErbB family kinases over other kinase families, and dose-dependently inhibited EGF-induced EGFR phosphorylation. Allitinib effectively inhibits the EGFR T790M/L858R double mutant with an IC 50 value of 12 nM. 177 – 181 Although enrolled in a phase II clinical trial ( NCT04671303 ) in December 2020 to evaluate the efficacy and safety of combined treatment with anlotinib in lung cancer, allitinib has not yet been administered. ES-072 ( 62 ) is a promising new generation of EGFR inhibitor, independently developed by Zhejiang Bossan Pharmaceutical Co Ltd, that is superior to the third-generation EGFR inhibitors. It is specifically designed to inhibit EGFR L858R/Del19 and EGFR T790M , while also addressing resistance acquired from first-generation EGFR inhibitors without T790M variants, as well as those from third-generation EGFR inhibitors. Notably, preclinical data indicates that ES-072 has the ability to penetrate the blood-brain barrier, making it a potentially effective treatment for brain metastases. In January 2018, ES-072 was registered for a phase I clinical trial (CTR20180074) to assess its efficacy in NSCLC patients with EGFR mutations. 182 This single-center, open, dose-escalation trial aims to evaluate the safety and tolerability of ES-072 in patients with locally advanced or metastatic NSCLC. Additionally, Bossan Pharmaceutical Co Ltd has collaborated with CBT Pharmaceuticals to develop combination therapies involving ES-072 and c-Met inhibitors, as well as PD-1 antibodies. Several clinical trial applications related to ES-072 have been accepted in recent years (CXHL1700078, CXHL1700080, CXHL1700079), and clinical trial approval documents have been obtained, highlighting the growing interest and potential of this promising drug. 183 YK-029A ( 63 ), an oral, irreversible third-generation EGFR TKI, is another osimertinib analog developed by Hainan Yuekang Biopharmaceutical Co Ltd. The drug is intended to treat advanced NSCLC with drug resistance and disease progression acquired by T790M gene mutation after previous treatment of EGFR TKIs. YK-029A has shown promise in preclinical studies, leading to its registration for a clinical phase I trial (CTR20180350) in May 2018. Furthermore, several clinical trial applications related to YK-029A have been accepted (CXHL2200062, CXHL2101515, CXHL1700173, CXHL1700174), and clinical trial approval documents have been obtained in recent years. These developments suggest growing interest in and potential for YK-029A as a treatment option for patients with advanced NSCLC. 184 , 185 Covalent EGFR inhibitors in terminated clinical trials With the emergence of new covalent EGFR inhibitors entering clinical trials, some clinical trials involving EGFR inhibitors have been terminated for various reasons. Canertinib (CI-1033; PD-183805) ( 64 ) is an irreversible inhibitor of the EGFR that effectively inhibits cellular EGFR and ErbB2 autophosphorylation with IC 50 s of 7.4 and 9 nM, respectively. In cultured melanoma cells (RaH3 and RaH5), canertinib significantly inhibits their growth in a dose-dependent manner, leading to G1-phase cell cycle arrest without inducing apoptosis. Notably, 1 μM canertinib also inhibits ErbB1-3 receptor phosphorylation and decreases Akt-, ERK1/2-, and Stat3 activity in both cell lines. 186 – 189 Canertinib was enrolled in clinical trials in December 2002, and completed studies investigating its efficacy in combination with paclitaxel/carboplatin for the first-line treatment of NSCLC ( NCT00174356 ), as well as in patients with metastatic (stage IV) breast cancer ( NCT00051051 ) and as a single agent for the treatment of advanced NSCLC ( NCT00050830 ) between 2002 and 2007. However, no further follow-up on canertinib has been reported since. Additionally, canertinib has shown potential as a treatment against vaccinia virus respiratory infection in mice. Rociletinib (Xegafri TM , CO-1686) ( 65 ), developed by Clovis Oncology, is a specific mutant agent for the treatment of NSCLC, belonging to the third-generation EGFR TKIs. 190 , 191 In the EGFR T790M , the anilinopyrimidine group in rociletinib forms two hydrogen bonds with Met793 amide and carbonyl backbone, which became a hydrophobic interaction in the T790M structure. Rociletinib was also able to form two hydrogen bonds in EGFR L858R . These include one between nitrogens in the pyrimidine group, and another between the fluoromethyl and Thr790. In both active (DFG-in/αC-in) conformations, the acrylamide group in rociletinib covalently binds to Cys797. 192 However, it induced various adverse reactions in clinical trials, including nausea (35%), fatigue (24%), diarrhea (22%), prolonged QT interval (22%) and hyperglycemia (22%). Hyperglycemia was mainly a tertiary adverse event, but it could be controlled by tapering or oral metformin. Despite this, at the April 2016 ODAC meeting, experts voted to delay approval of rociletinib, and Clovis announced the termination of rociletinib. 193 Naquotinib (ASP8273) ( 66 ) is an orally available, irreversible and mutant-selective EGFR L858R/T790M inhibitor that has shown potential as an antitumor agent. It covalently bound to an EGFR mutant (L858R/T790M) through cysteine residues to chronically inhibit phosphorylation of EGFR. Naquotinib also inhibits signaling pathways through ERK and Akt, and is active against EGFR mutant cell lines resistant to other EGFR TKIs such as AZD9291 and CO-1686. 194 , 195 In May 2017, Astellas announced the termination of a phase III clinical study ( NCT02588261 ) of naquotinib in NSCLC due to a recommendation from the Independent Data Monitoring Committee (IDMC). Subsequently, as a result, clinical trials of naquotinib have stopped recruiting patients altogether. Mavelertinib (PF-06747775) ( 67 ) is an orally available and irreversible EGFR TKI that selectively targets various EGFR mutants, such as Del, L858R, T790M/L858R and T790M/Del, with less than 50% effect or inhibition against all nonkinase targets. 196 – 198 In May 2015, a clinical study ( NCT02349633 ) was initiated to investigate mavelertinib in patients with NSCLC EGFR mutation (Del 19 or L858R +/− T790M). However, due to strategic reasons and changes in the external environment, the study was eventually discontinued in June 2021 when results were updated. Covalent EGFR inhibitors in preclinical research In addition, several covalent EGFR inhibitors are still in preclinical development. CL-387785 (EKI-785, WAY-EKI 785) ( 68 ) is a highly selective and irreversible EGFR inhibitor that specifically inhibits kinase activity of the protein (IC 50  = 370 pM). It effectively blocks autophosphorylation of receptors in EGF-stimulated cells (IC 50 approximately 5 nM) and inhibits cell proliferation in a cytostatic manner mainly in cell lines overexpressing EGFR or c-ErbB-2 (IC 50  = 31–125 nM). While most EGFR mutants transform cells and make them sensitive to erlotinib and gefitinib, the exon 20 insertion transformation confers resistance to these inhibitors but makes the cells more sensitive to the irreversible inhibitor, CL-387785. CL-387785 has also shown potential to overcome T790M mutation-related resistance at the functional level, possibly by effectively inhibiting downstream signaling pathways. 199 – 202 Despite its promising profile, no recent reports are available on CL-387785, as it remains under clinical development. Currently, there are also various lead compounds being developed as covalent EGFR inhibitors and antitumor agents. In 2009, Pasi et al. identified a series of covalent pyrimidine EGFR inhibitors, including WZ 3146, WZ 4002, and WZ 8040, through screening a library of irreversible kinase inhibitors specific to EGFR T790M . 203 These compounds showed a desirable 300-fold lower IC 50 against the PC9GR cells compared to clinical-stage inhibitors such as HKI-272. In vitro, they were 30-100 folds more effective against EGFR T790M and up to 100-fold less effective against WT-EGFR than quinazoline based EGFR inhibitors. Additionally, they have demonstrated efficacy in murine models of lung cancer driven by EGFR T790M . Among them, WZ 4002 ( 69 ) exhibited the highest efficacy and effectively inhibited the phosphorylation of EGFR, Akt, and ERK1/2. Some presentive promising molecules are listed in (Table 1 ). Four synthesized derivatives of 6- or 7-acrylamide-4-anilino-quinazolines, PD 160678 ( 70 ), PD 168393 ( 71 ), PD 160879 ( 72 ), PD 174265 ( 73 ), irreversibly inhibit EGFR TK activity with IC 50 values 0.45–0.70 nM. 204 Covalent p53 modulators P53 is a crucial protein that regulates the cell cycle and acts as a tumor suppressor. 205 Studies have shown that approximately half of all human cancers, including serous ovarian cancer, lung squamous cell cancer, lung small cell cancer, triple-negative breast cancer, and squamous esophageal cancer, have alterations in the p53 gene, resulting in a loss of p53 function or decreased p53 expression. 206 – 208 As a tumor suppressor TFs closely linked to PPIs, p53 plays a critical role in regulating gene expression, promoting tumor cell cycle arrest, apoptosis, and DNA repair. It can activate nearby or distant genes in response to an enhancer, while also indirectly inhibiting the transcription of numerous genes. 209 – 213 P53 can be categorized as mutant type or wild type, with mutant p53 promoting tumorigenesis and wild-type p53 having broad-spectrum tumor inhibition. 206 – 208 TP53 mutations typically reduce the expression of p53 protein or produce inactive variants, thus compromising its cancer-inhibiting properties. As a result, therapeutic strategies are needed to restore p53 function. However, most small molecules target overexpressed proteins by inhibiting their activity, making p53 an “undruggable” target. Covalent modulators directly targeting p53 In 2022, Kevan M. Shokat’s team continued their research and development work on the KRAS G12S mutant and developed a small molecule covalent inhibitor of p53-Y220C mutant, known as KG13 ( 74 ) (Table 1 ). 214 This inhibitor is specifically designed to bind to the p53 Y220C mutant, which restores the thermal stability of p53 protein to the level of wild-type p53 protein and activates the expression of downstream genes. The researchers designed 13 small molecule drugs to target the pocket structure formed by p53 Y220C in space. After a series of structural modifications and screening, KG13 was selected as the best small molecule compound with the highest covalent labeling rate and thermal stability recovery rate. Additionally, cells treated with KG13 demonstrated p53 Y220C-dependent p53 target gene activation, inhibition of cell growth, and increased caspase activity. Covalent p53-MDM2 PPI inhibitors Both Murine double minute 2 (MDM2, HMD2 in human) and MDMX act as negative regulators of p53, maintaining p53 at a low level by directly binding to its N-terminal and mediating its degradation in normal cells. 215 The primary mechanism of p53 degradation involves ubiquitylation by the E3 ubiquitin ligase MDM2, which leads to proteasomal degradation of p53. MDM2 amplification is frequently observed in several cancer types, especially in tumors that still retain wild-type p53. 216 , 217 Since MDM2-mediated ubiquitylation and degradation depend on its direct interaction with p53, researchers have been searching for small molecules that can inhibit this interaction to stabilize p53 and restore its activity. Although most p53-MDM2 inhibitors are non-covalent (which will be explained in the PPI inhibition part), some small molecule inhibitors that target p53-MDM2 have been found to be covalent, leading to the development of covalent p53-MDM2 inhibitors (Table 1 ). In 2017, Ishiba et al. performed mirror-image screening through D -proteins, which is an approach for identifying potential pharmaceutical candidates from homochiral resources, and revealed that NPD6878 (apomorphine) ( 75 ) was an MDM2–p53 inhibitor candidate with high potency. 218 At equipotent doses, R-(-)-apomorphine inhibited both the native L -MDM2- L -p53 interaction (IC 50  = 0.215 μM) and the mirror-image D -MDM2- D -p53 interaction (IC 50  = 0.195 μM). In addition, the enantiomer, S-(-)-apomorphine also showed equipotent inhibitory activity against the L -MDM2- L -p53 interaction (IC 50  = 0.175 μM). Among these, the achiral oxoapomorphine, which was converted from chiral apomorphine under aerobic conditions, served as the reactive species to form a covalent bond at Cys77 of MDM2 with Michael acceptors, leading to the inhibitory effect against the binding to p53. 219 In 2021, Hamachi et al. developed a small molecule covalent inhibitor, compound ( 76 ), based on the N -acyl- N -alkyl sulfonamide (NASA) reaction group, which can prevent the interaction between HDM2 and p53. 220 , 221 The researchers used a reactive NASA group as the warhead and conducted quality-based analysis to reveal the kinetics of covalent inhibition. They identified that the modification sites on HDM2 were the N-terminal alpha-amine and Tyr67. Using Nutlin-3 as a scaffold for a covalent inhibitor, the researchers found that Lys51, which an N ‑acyl‑ N ‑alkyl sulfonamide (NASA) warhead could target, was about 11 Å away from the 2-oxypiazine portion of Nutlin-3a. 222 , 223 They then structurally modified to generate a series of covalent compounds, which were tested for their ability to modify HDM2 in vitro. Through in vitro studies, this compound was found to exhibit severe p53-independent cytotoxicity, leading to its selection among the compounds. Additionally, it demonstrated a longer residence time on HDM2 compared to the non-covalent inhibitor Nutlin-3, resulting in higher HDM2/p53 inhibitory potency under diluted conditions. This compound was determined to selectively inhibit HDM2-induced p53 pathologically dependent apoptosis, instead of causing non-specific cytotoxicity caused by NASA warheads. This study marks a significant advancement in the rational design of effective covalent PPI inhibitors. Other covalent inhibitors Covalent Mcl-1 inhibitor Myeloid cell leukemia-1 (Mcl-1) is a crucial anti-apoptotic member of the Bcl-2 protein family that contributes significantly to the development of various human cancers. Targeting the BH3 binding groove of Mcl-1 has emerged as a promising approach for inhibiting its function and has become a focal point in the development of antitumor drugs. 224 – 227 In this regard, Lee et al. devised a drug design strategy based on a table of variable texture sites near the BH3 region opposite to the binding site. They utilized the covalent inhibitor, MAIM1 (77) (Table 1 ), which combines with Cys286 of the thalproquinone type, to effectively inhibit Mcl-1 activity (IC 50  = 450 nm). 228 This compound tightly binds to Mcl-1 and provides potential new pathways and drug precursor compounds for anti-apoptotic tumor therapy. Structural and functional analyses showed that the BH3 binding force and its inhibition of Bax were impaired by molecular bonding, as observed in the C286W mutagenesis simulation in vitro and in cells. This study offers valuable insights into the development of novel Mcl-1 inhibitors for cancer therapy. Covalent PKM2 inhibitor In a recent study on targeted covalent inhibitors, the Cross Center of Shanghai Institute of Organic Sciences and collaborators reported a novel PKM2 inhibitor, compound ( 78 ) (Table 1 ), based on trivalent arsine covalent warheads. 229 Although arsenic compounds are known for their toxicity and have been abandoned in modern medicine, a variety of organic arsine drugs targeting tumor delivery have been introduced into the clinic with success. 230 – 233 The trivalent arsine functional group has potential as a covalent warhead due to its ability to react with cysteine residues in proteins, affecting their activity and exerting a drug effect. Using organic arsenic covalent probes, chemical proteomics, and pharmacochemical methods, a highly active and specific covalent PKM2 inhibitor was developed. 229 The compound effectively inhibited ovarian cancer growth in vivo with IC 50 s value of 0.16 and 0.23 μM in PA-1 and A2780 cells, respectively. Treatment with this compound reduced tumor load in mice via gavage at 50 mg kg −1  day −1 . Its derivative compound formed a covalent bond with Cys474 near the allosteric activation pocket of PKM2, specifically inhibiting its activity without affecting PKM1. The study suggests that organic arsine compounds have potential as targeted covalent inhibitors, offering a broader application prospect in precision cancer therapy. Allosteric modulators The initial focus of rational drug design was on the orthosteric sites of therapeutic protein targets. 234 , 235 However, many of these targets have been found to be undruggable or difficult to target in their orthosteric sites due to their high affinity with substrates, lack of structural information, or high conservation of active sites. To overcome these challenges, allosteric regulation has been proposed as a strategy commonly used in nature to control cellular processes by modulating the affinity of biomolecules “at a distance”. Allosteric modulators can change the protein/substrate affinity in a highly predictable manner by stabilizing target proteins in an inactive or active state, leading to desirable controllability. 236 – 241 Allosteric modulators offer several advantages over orthosteric inhibitors. Firstly, allosteric ligands do not have to compete with high-affinity substrates, making it simpler to develop allosteric modulators. 238 , 242 , 243 Secondly, allosteric sites are diverse and confer better selectivity among homologous proteins, resulting in fewer side effects and greater value in clinical applications. 244 , 245 Thirdly, allosteric modulators have a desirable “ceiling of effect”. Once allosteric sites are occupied, no additional effects can be observed, indicating drug safety under overdose conditions. 246 Additionally, undruggable proteins can be targeted simultaneously by orthosteric inhibitors and allosteric modulators to achieve a synergistic effect and overcome resistance. Allosteric modulators can not only inhibit targets like orthosteric inhibitors but can also stabilize them or competitively occupy them if needed to improve pathological states. 6 , 47 , 247 According to their effects on the receptor, allosteric modulators can be classified into three categories: positive allosteric modulators (PAMs), which improve the action of orthosteric effectors but have no intrinsic activity; negative allosteric modulators (NAMs), which inhibit the function of orthosteric effectors; and silent allosteric modulators (SAMs), also known as neutral allosteric modulators, which inhibit allosteric activities by blocking the allosteric site of both PAMs and NAMs. 248 , 249 Therefore, the identification of allosteric sites and corresponding drug design has opened up new therapeutic opportunities for proteins that were previously considered “undruggable” or “difficult to target” at their orthosteric site. Since the concept of allosteric modulation was first proposed in the 1960s, a number of allosteric drugs have been applied in clinical practice, evaluated at clinical trial phases or preclinical stages. Initially, allosteric drug design focused on inhibiting kinases and GPCRs with highly conserved active sites, as an alternative option to overcome the undesired selectivity profiles and resistance that occurs in the clinical application of orthosteric modulators. 250 – 252 After a decade of development, the range of target categories has expanded to others, including several undruggable proteins that lack marketed drugs, such as KRAS and SHP2. It is noteworthy that some targets provide the opportunity for combined application of covalency and allostery in drug design. For instance, AMG510 (sotorasib), the first marketed KRAS inhibitor that was granted accelerated approval (Lumakras™, Amgen, Inc.) by the FDA, is a covalent allosteric inhibitor of KRAS G12C mutant, highlighting the significance of rational design of covalent allosteric drugs. 55 In this part, we summarize the development in drug design and clinic trials targeting allosteric sites of undruggable proteins and those proteins which are hard to selectively target with orthosteric inhibitors (Table 2 and Fig. 2 ). Table 2 Allosteric modulators targeting undruggable proteins Compound name and structure Target Cancer cell line (activity) Indications Status/clinical trial identifier Ref. MRTX-1133 ( 79 ) KRAS G12D – Colorectal cancer, NSCLC, pancreas cancer Ongoing NCT05737706 (I/II) 254 TNO-155 ( 80 ) SHP2 – Colorectal cancer, esophageal cancer, NSCLC, etc. Ongoing NCT05541159 (I), NCT05490030 (I), NCT04000529 (I), NCT03114319 (I), NCT04330664 (I/II) 272 JAB-3068 ( 81 ) a SHP2 – Esophagus cancer, NSCLC, etc. Ongoing NCT04721223 (I/II), NCT03565003 (I/II), NCT03518554 (NA) 274 RMC-4630 ( 82 ) SHP2 – Colorectal cancer, NSCLC Completed NCT03989115 (I/II) Ongoing NCT04916236 (I), NCT03634982 (I), NCT05054725 (II) 275 JAB-3312 ( 83 ) a SHP2 – Colorectal cancer, esophagus tumor, NSCLC, etc. Ongoing NCT05288205 (I/II), NCT04720976 (I/II), NCT04121286 (I), NCT04045496 (I) 277 RLY-1971 ( 84 ) a SHP2 – Advanced solid tumor Completed NCT04252339 (I) 278 BBP-398 ( 85 ) SHP2 – Metastatic NSCLC Ongoing NCT05621525 (I), NCT05480865 (I), NCT05375084 (I), NCT04528836 (I) 279 ERAS-601 ( 86 ) a SHP2 – Acute myelogenous leukemia, NSCLC Ongoing NCT04959981 (I/II), NCT04866134 (I/II), NCT04670679 (I) 280 SH3809 ( 87 ) a SHP2 – Advanced solid tumor Ongoing NCT04843033 (I) 281 ET-0038 ( 88 ) a SHP2 – Advanced solid tumor Ongoing NCT05354843 (I), NCT05525559 (I) 282 ICP-189 ( 89 ) a SHP2 – Advanced solid tumor Ongoing NCT05370755 (I) 283 SHP099 ( 90 ) SHP2 Caco-2 (IC 50  = 0.07 μM) – Preclinical 286 RMC-4550 ( 91 ) SHP2 MIA PaCa-2, NCI-H35 (IC 50  = 0.583 nM) – Preclinical 289 PCC0208023 ( 92 ) SHP2 LS180, HCT116 (IC 50  = 2.1 nM) – Preclinical 287 TK-453 ( 93 ) SHP2 – – Preclinical 293 Cinacalcet (AMG-073) ( 94 ) CaS – Hypercalcemia, hyperparathyroidism, SHPT Marketed 657 Maraviroc ( 95 ) CCR5 – HIV infection Marketed 658 Ticagrelor (AZD-6140) ( 96 ) P2Y12 – Arterial thrombosis, Ischemic stroke, etc. Marketed 659 Avacopan (CCX168) ( 97 ) C5a1 – Vasculitis Marketed 660 Vercirnon ( 98 ) CCR9 – Celiac disease, inflammatory bowel disease Completed NCT01277666 (III), NCT00102921 (II), NCT01114607 (I), etc. Terminated NCT01536418 (III), NCT01318993 (III), etc. 661 Mavoglurant ( 99 ) mGlu5 – Cocaine addiction, obsessive–compulsive disorder Completed NCT02920892 (II), etc. Ongoing NCT03327792 (I); NCT05203965 (0) Withdrawn NCT04771143 (I) Terminated NCT01019473 (II), etc. 662 T-62 ( 100 ) A1 – Neuropathic pain, postherpetic neuralgia Withdrawn NCT00506610 (II) Terminated NCT00809679 (II) 663 AZD-8529 ( 101 ) a mGlu2 – Schizophrenia Completed NCT02401022 (II), NCT00921804 (II), etc. 664 Raseglurant ( ADX10059 ) ( 102 ) mGlu5 – Gastroesophageal reflux, migraine, Parkinson’s disease Completed NCT00820079 (II), NCT00810485 (II) Terminated NCT00820105 (II) 665 Basimglurant (RG-7090) ( 103 ) mGlu5 – Trigeminal neuralgia Completed NCT02433093 (I) Ongoing NCT05059327 (II), NCT05217628 (II) 666 JNJ-40411813 ( 104 ) mGlu2 – Epilepsy Completed NCT01582815 (II), NCT01323205 (II), NCT04677530 (I), etc. Ongoing NCT04836559 (II) 667 (11 C) JNJ-42491293 ( 105 ) a mGlu2 – Psychiatric disorder Completed NCT01359852 (I) 668 MK-7622 ( 106 ) M1 – Alzheimer’s disease Terminated NCT01852110 (II) 669 RG-7342 ( 107 ) a mGlu5 – Schizophrenia Terminated NCT02196636 (I) 670 ODM-106 ( 108 ) a GABAB – Essential tremor Completed NCT02393950 (I) 671 JNJ-55375515 ( 109 ) mGlu2 – Neurological disease, psychiatric disorder Completed NCT03405441 (I), NCT02623491 (I) 672 MK-6884 ( 110 ) M4 – Alzheimer’s disease Completed NCT02621606 (I) 673 ASP-4345 ( 111 ) a D1 – Cognitive disorder Completed NCT03557931 (II), NCT02720263 (I) 674 TAK-071 ( 112 ) M1 – Cognitive disorder, Parkinson’s disease Ongoing NCT04334317 (II) Terminated NCT02918266 (I), NCT02769065 (I) 675 Foliglurax (DT-1687) ( 113 ) mGlu4 – Parkinson’s disease Completed NCT03162874 (II), etc. Withdrawn NCT03331848 (II) Terminated NCT04322227 (I), etc. 676 ASP-8302 ( 114 ) a M3 – Urinary dysfunction Completed NCT03702777 (II), NCT03361540 (I) 677 HTL0014242 (TMP-301) ( 115 ) mGlu5 – Neurological disease, psychiatric disorder Completed NCT04462263 (I), NCT03785054 (I) 678 JNJ-2463 (nimacimab) ( 116 ) a CB1 – Diabetic gastroparesis Unknown status NCT03900325 (II) 679 RGH-618 ( 117 ) mGlu5 – Generalized anxiety disorder No progress 680 Data collected from https://clinicaltrials.gov [last accessed March 2023] a The chemical formula was not disclosed Fig. 2 Allosteric inhibitors targeting undruggable proteins. Allosteric modulators change the protein/substrate affinity by stabilizing target proteins in an inactive or active state “at a distance”. a Binding mode of selected allosteric modulators: RAS allosteric inhibitors interact with mutant amino acids in switch II region to induce conformational changes, thereby locking KRAS in an inactive conformation; SHP2 allosteric inhibitors directly stabilize the autoinhibited conformation of SHP2, thereby preventing interactions between the catalytic PTP domain and SHP2 substrates; GPCR allosteric inhibitors can be classified into PAMs, allosteric antagonists and NAMs based on their mode of action. b Map of marketed, clinical and preclinical allosteric inhibitors in signaling pathways RAS allosteric inhibitors The interactions of KRAS-GTP or KRAS-GDP are closely linked to the activated state of KRAS and subsequently affect its signal transmissions. In general, the inactive KRAS conformation that is involved in KRAS-GDP binding is preferred. 43 Structural biology analysis has identified two switches, switch I and switch II, on the surface of the KRAS protein that change their status based on the binding status of KRAS. The switch II region is particularly significant because of its high conformational variability, which provides an entry point for allosteric regulation. Allosteric inhibitors that interact with mutant amino acids in switch II region can induce conformational changes, resulting in a more inactive KRAS conformation. Thus, the development of allosteric inhibitors that specifically target KRAS and inhibit its abnormal function presents a promising approach to target KRAS mutants. 253 The advantages of irreversibility provided by covalent bonding have made covalent inhibitors an effective approach to inhibiting RAS mutations. In fact, the success of RAS inhibitors provides a classic example of covalent inhibitors as well as allosteric modulators. Most of the covalent KRAS inhibitors mentioned earlier, such as the approved Sotorasib (AMG510) and Adagrasib (MRTX849), achieve inhibitory effects by stabilizing the conformation of KRAS G12C mutants in an inactive state through covalent binding to residues in the allosteric site. This highlights the significance of covalent inhibitors and allosteric regulation in drug discovery, particularly in targeting KRAS-related diseases. KRAS G12C allosteric inhibitors The KRAS G12C mutant protein contains a mutant cysteine, Cys12, which provides a potential covalent site for inhibitors. When an inhibitor with a covalent warhead binds covalently to the mutant cys12, it induces a new allosteric pocket, S-II P, in the switch II region. The small molecule inhibitor then interacts with the corresponding amino acid, resulting in a conformational change of the KRAS protein. These effects reduce the affinity between GTP and KRAS, prevent GDP from being replaced by GTP through GEF catalysis, and ultimately lock KRAS mutants in an inactive state. 51 Based on this action site, several small-molecule inhibitors targeting KRAS have been developed, almost all of which are covalent and were discussed in the previous chapter. These include Sotorasib (AMG-510), Adagrasib (MRTX-849), ARS-853, ARS-1620, LY-3537982, GDC-6036 (RG-6300), D-1553, ARS-3248 (JNJ-74699157), JDQ-443, and SML series compounds, among others, such as LY-3537982, ARS-853, ARS-1620, and 6H05 series compounds (Table 2 ). KRAS G12D allosteric inhibitors KRAS G12D is a more prevalent KRAS mutation type that is found in various cancers, including pancreatic cancer, colorectal cancer, and lung adenocarcinoma. As such, it is a potential target for the development of selective KRAS mutation inhibitors. However, effectively targeting other KRAS mutants presents several challenges that must be overcome. Unlike KRAS G12C , KRAS G12D lacks an active residue near the switch II binding pocket, which prevents the protein from undergoing covalent modification. Therefore, new approaches are required to design selective inhibitors with high affinity and drug potency for KRAS G12D and other non-C mutant KRAS mutations. Mirati Therapeutics has identified and characterized a selective, non-covalent, high-affinity KRAS G12D inhibitor, known as MRTX-1133 ( 79 ) (Table 2 ). 254 The inhibitor binds to the inactive form of KRAS G12D with an IC 50  < 2 nM, demonstrating an approximately 700-fold selectivity compared to KRAS WT . MRTX-1133 also inhibits the binding of RAF-RAS binding domain peptides to the active form of KRAS G12D with an IC 50 of 9 nM, and induces conformational changes in switch I and switch II regions of KRAS protein. 254 By interacting with aspartic acid Asp12 and glutamic acid Glu62 in the switch II region of KRAS protein, MRTX-1133 plays an allosteric role, resulting in conformational changes of KRAS protein and inhibition of the KRAS signaling pathway in cells and tumor environments containing KRAS G12D mutations, thereby achieving an antitumor effect. In cell studies, MRTX-1133 exhibited a concentration-dependent inhibition of key KRAS pathway signaling molecules in KRAS G12D mutated HPAC (pancreatic cancer) and GP2D (colorectal cancer) cell lines, including the phosphorylation of extracellular signal-regulated kinase 1/2 (pERK), the phosphorylation of S6 (pS6), the phosphorylation of 4EBP1 (p4EBP1), and the expression of dual specificity phosphatase 4 or 6 (DUSP4/6). Besides, MRTX-1133 inhibited KRAS-dependent signaling and promoted tumor regression in xenograft models. 255 – 258 SHP2 allosteric inhibitors Src homology 2-containing protein tyrosine phosphatase 2 (SHP2) is a non-receptor protein tyrosine phosphatase (PTP) encoded by PTPN11 gene. 259 As protein tyrosine phosphorylation plays an essential role in multiple intracellular processes, SHP2 is involved in the regulation of multiple signaling pathways, including those involved in cancer cells, such as RAS-MAPK, PI3K-AKT and JAK-STAT pathways. Besides, SHP2 is related to some functions of PD-1/PD-L1, thus playing a role in the regulation of immune system. 260 – 264 In addition, SHP2 overexpression or activation can mediate drug resistance in various cancers, including leukemia, non-small cell carcinoma, and breast cancer. Therefore, SHP2 has been considered a potential therapeutic target for cancer therapy. 265 – 268 Structurally, SHP2 contains two SH2 domains (N-SH2 and C-SH2) at the N-terminal, a catalytic PTP domain, and two phosphorylable tyrosine residues (Tyr542 and Tyr580) at the C-terminal. Typically, the interaction between the N-SH2 domain and the PTP domain leads to an auto-inhibited closed conformation of the SHP2 protein. 260 , 269 Upon stimulation stimulated by growth factors or cytokines, the SHP2 protein is activated, exposing the catalytic PTP domain, due to the occupation of SH2 domain thus the blocking of N-SH2-PTP interaction. As a consequence, the catalytic site of SHP2 is available to its substrates, and subsequent signal transductions could be activated. Initially, attempts to regulate SHP2 focus on identifying conventional competitive inhibitors specific to the PTP domain, also known as orthosteric inhibitors. Some