Covalent Epidermal Growth Factor Receptor Research Articles

Covalent inhibition of epidermal growth factor receptor using a long-lived iridium(III)-afatinib probe

The overexpression and overactivation of epidermal growth factor receptor (EGFR) are frequently observed in human cancers, including squamous cell carcinoma and adenocarcinoma. In this study, a covalent EGFR probe was developed by conjugating afatinib to an iridium(III) scaffold. Complex 1 showed enhanced luminescence in living epidermoid squamous carcinoma A431 cells compared to other cell lines, via engaging EGFR as confirmed via CETSA and knockdown experiments. Moreover, complex 1 inhibited downstream targets of EGFR in cellulo with repression persisting after removal of the complex, indicating an irreversible mode of inhibition. Finally, complex 1 showed potent antiproliferative activity against A431 cells with comparable potency to afatinib alone. To our knowledge, complex 1 is the first EGFR covalent inhibitor based on an iridium scaffold reported in the literature, with the potential to be further explored as a theranostic agent in the future.

Pitfalls and Considerations in Determining the Potency and Mutant Selectivity of Covalent Epidermal Growth Factor Receptor Inhibitors.

Enzyme inhibitors that form covalent bonds with their targets are being increasingly pursued in drug development. Assessing their biochemical activity relies on time-dependent assays, which are distinct and more complex compared with methods commonly employed for reversible-binding inhibitors. To provide general guidance to the covalent inhibitor development community, we explored methods and reported kinetic values and experimental factors in determining the biochemical activity of various covalent epidermal growth factor receptor (EGFR) inhibitors. We showcase how liquid handling and assay reagents impact kinetic parameters and potency interpretations, which are critical for structure-kinetic relationships and covalent drug design. Additionally, we include benchmark kinetic values with reference inhibitors, which are imperative, as covalent EGFR inhibitor kinetic values are infrequently consistent in the literature. This overview seeks to inform best practices for developing new covalent inhibitors and highlight appropriate steps to address gaps in knowledge presently limiting assay reliability and reproducibility.

Osimertinib Covalently Binds to CD34 and Eliminates Myeloid Leukemia Stem/Progenitor Cells.

Osimertinib binds CD34 and selectively kills CD34+ leukemia cells to induce remission in preclinical models and patients with AML with a high percentage of CD34+ blasts, providing therapeutic options for myeloid leukemia patients.

Targeting Cytotoxic Agents through EGFR-Mediated Covalent Binding and Release.

A major drawback of cytotoxic chemotherapy is the lack of selectivity toward noncancerous cells. The targeted delivery of cytotoxic drugs to tumor cells is a longstanding goal in cancer research. We proposed that covalent inhibitors could be adapted to deliver cytotoxic agents, conjugated to the β-position of the Michael acceptor, via an addition-elimination mechanism promoted by covalent binding. Studies on model systems showed that conjugated 5-fluorouracil (5FU) could be released upon thiol addition in relevant time scales. A series of covalent epidermal growth factor receptor (EGFR) inhibitors were synthesized as their 5FU derivatives. Achieving the desired release of 5FU was demonstrated to depend on the electronics and geometry of the compounds. Mass spectrometry and NMR studies demonstrated an anilinoquinazoline acrylate ester conjugate bound to EGFR with the release of 5FU. This work establishes that acrylates can be used to release conjugated molecules upon covalent binding to proteins and could be used to develop targeted therapeutics.

Addressing the Osimertinib Resistance Mutation EGFR-L858R/C797S with Reversible Aminopyrimidines.

Drug resistance mutations emerging during the treatment of non-small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) inhibitors represent a major challenge in personalized cancer treatment and require constant development of new inhibitors. For the covalent irreversible EGFR inhibitor osimertinib, the predominant resistance mechanism is the acquired C797S mutation, which abolishes the covalent anchor point and thus results in a dramatic loss in potency. In this study, we present next-generation reversible EGFR inhibitors with the potential to overcome this EGFR-C797S resistance mutation. For this, we combined the reversible methylindole-aminopyrimidine scaffold known from osimertinib with the affinity driving isopropyl ester of mobocertinib. By occupying the hydrophobic back pocket, we were able to generate reversible inhibitors with subnanomolar activity against EGFR-L858R/C797S and EGFR-L858R/T790M/C797S with cellular activity on EGFR-L858R/C797S dependent Ba/F3 cells. Additionally, we were able to resolve cocrystal structures of these reversible aminopyrimidines, which will guide further inhibitor design toward C797S-mutated EGFR.

Design, synthesis, and biological evaluation of 2-arylamino-4-(piperidin-4-yloxy)pyrimidines as potent EGFRT790M/L858R inhibitors to treat non-small cell lung cancer

Three types of 2-arylamino-4-(piperidin-4-yloxy)pyrimidines (I–III) were designed and synthesized as covalent EGFR(epidermal growth factor receptor)T790M/L858R inhibitors by replacement of the common reported 4-(3-amino)phenoxyl moiety with 4-(4-hydroxy)piperidine-4-oxyl, and the introduction of fused-thiophene or -pyrrole on the pyrimidine core to strategically achieve conformational restriction. According to our biological evaluation, it was found that compound 9i could potently suppress EGFRT790M/L858R kinase and H1975 cell proliferation, with IC50 values of 4.902 nM and 0.6210 μM, respectively. Further study showed that 9i not only demonstrated highly selective inhibitory effects toward EGFRT790M/L858R over wild-type EGFR (EGFRWT), but it also had low cytotoxicity against normal HBE(human bronchial epithelial) and L-02 cells. Action mechanism studies showed that 9i effectively hindered cell migration and promoted apoptosis by AO(Acridine Orange)/EB(Ethidium Bromide) staining. These data would provide important clues for the screening of novel covalent EGFRT790M/L858R inhibitors for non-small cell lung cancer (NSCLC) treatment.

Advances in covalent drug discovery.

Covalent drugs have been used to treat diseases for more than a century, but tools that facilitate the rational design of covalent drugs have emerged more recently. The purposeful addition of reactive functional groups to existing ligands can enable potent and selective inhibition of target proteins, as demonstrated by the covalent epidermal growth factor receptor (EGFR) and Bruton’s tyrosine kinase (BTK) inhibitors used to treat various cancers. Moreover, the identification of covalent ligands through ‘electrophile-first’ approaches has also led to the discovery of covalent drugs, such as covalent inhibitors for KRAS(G12C) and SARS-CoV-2 main protease. In particular, the discovery of KRAS(G12C) inhibitors validates the use of covalent screening technologies, which have become more powerful and widespread over the past decade. Chemoproteomics platforms have emerged to complement covalent ligand screening and assist in ligand discovery, selectivity profiling and target identification. This Review showcases covalent drug discovery milestones with emphasis on the lessons learned from these programmes and how an evolving toolbox of covalent drug discovery techniques facilitates success in this field.

Discovery of Potent PROTACs Targeting EGFR Mutants through the Optimization of Covalent EGFR Ligands.

Drug resistance caused by epidermal growth factor receptor (EGFR) mutation has largely limited the clinical use of EGFR tyrosine kinase inhibitors (EGFR-TKIs) for the treatment of non-small-cell lung cancer (NSCLC). Herein, to overcome the intractable problem of drug resistance, proteolysis targeting chimeras (PROTACs) targeting EGFR mutants were developed by optimizing covalent EGFR ligands. Covalent or reversible covalent pyrimidine- or purine-containing PROTACs were designed, synthesized, and evaluated. As a consequence, covalent PROTAC CP17, with a novel purine-containing EGFR ligand, was discovered as a highly potent degrader against EGFRL858R/T790M and EGFRdel19, reaching the lowest DC50 values among all reported EGFR-targeting PROTACs. Furthermore, CP17 exhibited excellent cellular activity against the H1975 and HCC827 cell lines with high selectivity. Mechanism investigation indicated that the lysosome was involved in the degradation process. Importantly, the covalent binding strategy was proven to be an effective approach for the design of PROTACs targeting EGFRL858R/T790M, which laid the practical foundation for further development of potent EGFR-targeting PROTACs.

Discovery of novel potent covalent inhibitor-based EGFR degrader with excellent in vivo efficacy

Although several Epidermal growth factor receptor (EGFR) inhibitors have been approved for the treatment of non-small-cell lung cancers (NSCLC), acquired drug resistance and side effects largely encumbered their application in clinic. The emerging technology Proteolysis targeting chimera (PROTAC) could be an alternative strategy to overcome these problems. Here, we reported the discovery of Dacomitinib-based EGFR degraders. Promising compound 13 can effectively induce degradation of EGFRdel19 with DC50 value of 3.57 nM in HCC-827 cells, but not to other EGFR mutant, wild-type EGFR protein and the same family receptors (HER2 and HER4). Of note, 13 is the first EGFR-PROTAC to evaluate antitumor effect in vivo, and exhibited excellent antitumor efficacy (TGI = 90%) at a dose of 30 mg/kg without causing observable toxic effects. The preliminary mechanism study demonstrated that 13 can efficiently induce EGFR protein degradation through ubiquitin proteasome pathway and inhibit phosphorylation of downstream pathways in vitro and in vivo, which indicated that 13 exerted antitumor effect by degradation of EGFR protein in tumor tissue. Overall, our study provided further evidence to validate EGFR-PROTACs as a promising strategy for lung cancer therapy.

Discovery and optimization of covalent EGFR T790M/L858R mutant inhibitors

Epidermal growth factor receptor (EGFR) inhibitors have clinical utility in the treatment of non-small cell lung cancer (NSCLC) patients. Despite encouraging clinical efficacy with these agents, many patients develop resistance due to sensitizing (or activating) mutations ultimately leading to disease progression. In the majority of the cases, this resistance is due to the T790M mutation and frequently coexisting L858R. In addition, EGFR wild type receptor inhibition can lead to on target related dose limiting toxicities such as rash and diarrhea. We describe herein the identification of a mutant selective lead compound 12, an irreversible covalent inhibitor of EGFR T790M/L858R resistance mutations with selectivity over the wild type form. Significant tumor growth inhibition in preclinical models was observed with this lead.

Potent and Selective Inhibitors of the Epidermal Growth Factor Receptor to Overcome C797S-Mediated Resistance.

The epidermal growth factor receptor (EGFR) harboring activating mutations is a clinically validated target in non-small-cell lung cancer, and a number of inhibitors of the EGFR tyrosine kinase domain, including osimertinib, have been approved for clinical use. Resistance to these therapies has emerged due to a variety of molecular events including the C797S mutation which renders third-generation C797-targeting covalent EGFR inhibitors considerably less potent against the target due to the loss of the key covalent-bond-forming residue. We describe the medicinal chemistry optimization of a biochemically potent but modestly cell-active, reversible EGFR inhibitor starting point with sub-optimal physicochemical properties. These studies culminated in the identification of compound 12 that showed improved cell potency, oral exposure, and in vivo activity in clinically relevant EGFR-mutant-driven disease models, including an Exon19 deletion/T790M/C797S triple-mutant mouse xenograft model.

Untangling Dual-Targeting Therapeutic Mechanism of Epidermal Growth Factor Receptor (EGFR) Based on Reversed Allosteric Communication.

Dual-targeting therapeutics by coadministration of allosteric and orthosteric drugs is drawing increased attention as a revolutionary strategy for overcoming the drug-resistance problems. It was further observed that the occupation of orthosteric sites by therapeutics agents has the potential to enhance allosteric ligand binding, which leads to improved potency of allosteric drugs. Epidermal growth factor receptor (EGFR), as one of the most critical anti-cancer targets belonging to the receptor tyrosine kinase family, represents a quintessential example. It was revealed that osimertinib, an ATP-competitive covalent EGFR inhibitor, remarkably enhanced the affinity of a recently developed allosteric inhibitor JBJ-04-125-02 for EGFRL858R/T790M. Here, we utilized extensive large-scale molecular dynamics simulations and the reversed allosteric communication to untangle the detailed molecular underpinning, in which occupation of osimertinib at the orthosteric site altered the overall conformational ensemble of EGFR mutant and reshaped the allosteric site via long-distance signaling. A unique intermediate state resembling the active conformation was identified, which was further stabilized by osimertinib loading. Based on the allosteric communication pathway, we predicted a novel allosteric site positioned around K867, E868, H893, and K960 within the intermediate state. Its correlation with the orthosteric site was validated by both structural and energetic analysis, and its low sequence conservation indicated the potential for selective targeting across the human kinome. Together, these findings not only provided a mechanistic basis for future clinical application of the dual-targeting therapeutics, but also explored an innovative perception of allosteric inhibition of tyrosine kinase signaling.

Targeting EGFRL858R/T790M and EGFRL858R/T790M/C797S resistance mutations in NSCLC: Current developments in medicinal chemistry.

Both the first-generation reversible epidermal growth factor receptor (EGFR) inhibitors gefitinib and erlotinib and the second-generation covalent epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) afatinib have significantly improved the survival of non-small-cell lung cancer (NSCLC) patients with activating EGFR mutations. However, a secondary EGFRT790M mutation leads to the clinically acquired resistance to the first- and second-generation EGFR-TKIs drugs. A number of the third-generation wild-type sparing EGFR inhibitors, for example, WZ4002, CO1686, AZD9291, HM61713, EGF816, ASP8173, and PF0674775, have been developed, among which AZD9291 has been approved by US FDA for the treatment of NSCLC patients with EGFRT790M . More recently, a tertiary EGFRC797S mutation was reported as the dominant resistance mechanism to the third-generation irreversible inhibitors. It is highly desirable to develop the fourth-generation EGFR inhibitors. This review summarizes the mechanisms of acquired resistance and the latest medicinal chemistry advances on the third- and fourth-generation EGFR inhibitors, with special attention being paid to the allosteric and reversible inhibitors combating the tertiary EGFRC797S mutation.

Structure-Guided Development of Covalent and Mutant-Selective Pyrazolopyrimidines to Target T790M Drug Resistance in Epidermal Growth Factor Receptor.

Reversible epidermal growth factor receptor (EGFR) inhibitors prompt a beneficial clinical response in non-small cell lung cancer patients who harbor activating mutations in EGFR. However, resistance mutations, particularly the gatekeeper mutation T790M, limit this efficacy. Here, we describe a structure-guided development of a series of covalent and mutant-selective EGFR inhibitors that effectively target the T790M mutant. The pyrazolopyrimidine-based core differs structurally from that of aminopyrimidine-based third-generation EGFR inhibitors and therefore constitutes a new set of inhibitors that target this mechanism of drug resistance. These inhibitors exhibited strong inhibitory effects toward EGFR kinase activity and excellent inhibition of cell growth in the drug-resistant cell line H1975, without significantly affecting EGFR wild-type cell lines. Additionally, we present the in vitro ADME/DMPK parameters for a subset of the inhibitors as well as in vivo pharmacokinetics in mice for a candidate with promising activity profile.

Discovery of a Noncovalent, Mutant-Selective Epidermal Growth Factor Receptor Inhibitor.

Inhibitors targeting the activating mutants of the epidermal growth factor receptor (EGFR) have found success in the treatment of EGFR mutant positive non-small-cell lung cancer. A secondary point mutation (T790M) in the inhibitor binding site has been linked to the acquired resistance against those first generation therapeutics. Herein, we describe the lead optimization of a series of reversible, pan-mutant (L858R, del746-750, T790M/L858R, and T790M/del746-750) EGFR inhibitors. By use of a noncovalent double mutant (T790M/L858R and T790M/del746-750) selective EGFR inhibitor (2) as a starting point, activities against the single mutants (L858R and del746-750) were introduced through a series of structure-guided modifications. The in vitro ADME-PK properties of the lead molecules were further optimized through a number of rational structural changes. The resulting inhibitor (21) exhibited excellent cellular activity against both the single and double mutants of EGFR, demonstrating target engagement in vivo and ADME-PK properties that are suitable for further evaluation. The reversible, noncovalent inhibitors described complement the covalent pan-mutant EGFR inhibitors that have shown encouraging results in recent clinical trials.

ChemInform Abstract: Covalent EGFR Inhibitors: Binding Mechanisms, Synthetic Approaches, and Clinical Profiles

AbstractReview: 100 refs.

Covalent EGFR Inhibitors: Binding Mechanisms, Synthetic Approaches, and Clinical Profiles.

Being overexpressed in several types of cancer, the epidermal growth factor receptor (EGFR) is considered one of the key therapeutic targets in oncology. Although many first-generation EGFR inhibitors had been FDA approved for the treatment of certain types of cancer, patients soon developed resistance to these reversible ATP competitive inhibitors via mutations in the kinase domain of EGFR. A new trend was adopted to design covalent irreversible inhibitors, that is, second- and third-generation inhibitors. Second-generation inhibitors can inhibit the mutant forms but, unfortunately, they had dose limiting side effects due to wild-type EGFR inhibition. Third-generation inhibitors emerged shortly, which were capable of inhibiting the mutant forms exclusively while sparing the wild type. Many other strategies have also been developed to reduce the risk of covalent interactions with off-targets, thus improving the pharmacokinetic and/or pharmacodynamic profile of the antiproliferative agents. In this review, we focused mainly on second- and third-generation EGFR inhibitors, their binding mechanisms (either docking studies or co-crystallized structures), their synthetic approaches, clinical profiles, and limitations.

ChemInform Abstract: Recent Progress on Third Generation Covalent EGFR Inhibitors

AbstractReview: 44 refs.

The safety and efficacy of osimertinib for the treatment of EGFR T790M mutation positive non-small-cell lung cancer

ABSTRACTFirst- and second-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are the evidence-based first-line treatment for metastatic non-small-cell lung cancers (NSCLCs) that harbor sensitizing EGFR mutations (i.e. exon 19 deletions or L858R). However, acquired resistance to EGFR TKI monotherapy occurs invariably within a median time frame of one year. The most common form of biological resistance is through the selection of tumor clones harboring the EGFR T790M mutation, present in >50% of repeat biopsies. The presence of the EGFR T790M mutation negates the inhibitory activity of gefitinib, erlotinib, and afatinib. A novel class of third-generation EGFR TKIs has been identified by probing a series of covalent pyrimidine EGFR inhibitors that bind to amino-acid residue C797 of EGFR and preferentially inhibit mutant forms of EGFR versus the wild-type receptor. We review the rapid clinical development and approval of the third-generation EGFR TKI osimertinib for treatment of NSCLCs with EGFR-T790M.

Redox regulation of epidermal growth factor receptor signaling during the development of pulmonary hypertension

The development of pulmonary hypertension (PH) involves the uncontrolled proliferation of pulmonary smooth muscle cells via increased growth factor receptor signaling. However, the role of epidermal growth factor receptor (EGFR) signaling is controversial, as humans with advanced PH exhibit no changes in EGFR protein levels and purpose of the present study was to determine whether there are post-translational mechanisms that enhance EGFR signaling in PH.The EGFR inhibitor, gefinitib, significantly attenuated EGFR signaling and prevented the development of PH in monocrotaline (MCT)-exposed rats, confirming the contribution of EGFR activation in MCT induced PH. There was an early MCT-mediated increase in hydrogen peroxide, which correlated with the binding of the active metabolite of MCT, monocrotaline pyrrole, to catalase Cys377, disrupting its multimeric structure. This early oxidative stress was responsible for the oxidation of EGFR and the formation of sodium dodecyl sulfate (SDS) stable EGFR dimers through dityrosine cross-linking. These cross-linked dimers exhibited increased EGFR autophosphorylation and signaling. The activation of EGFR signaling did not correlate with pp60src dependent Y845 phosphorylation or EGFR ligand expression. Importantly, the analysis of patients with advanced PH revealed the same enhancement of EGFR autophosphorylation and covalent dimer formation in pulmonary arteries, while total EGFR protein levels were unchanged. As in the MCT exposed rat model, the activation of EGFR in human samples was independent of pp60src phosphorylation site and ligand expression. This study provides a novel molecular mechanism of oxidative stress stimulated covalent EGFR dimerization via tyrosine dimerization that contributes into development of PH.