Non-covalent Small Molecule Inhibitors Research Articles

Abstract 7264: OPN-9840, a non-covalent potent pan-TEAD inhibitor, exhibits single agent efficacy in preclinical malignant mesothelioma models

Abstract The Hippo pathway regulates critical cellular processes during development. In ~10% of human cancers, Hippo pathway dysregulation results in YAP/TAZ overactivation and increased TEAD-dependent transcription that promotes tumor growth and therapy resistance - often by supporting drug-tolerant persister survival. In 40% of malignant mesotheliomas (MM), neurofibromatosis 2 (NF2) gene mutations inactivate the Merlin protein, an upstream negative regulator of TEAD. Preclinical studies indicate high TEAD dependency in NF2-mutant MM. We have discovered an array of potent covalent and non-covalent small molecule inhibitors that occupy the palmitate-binding pocket of TEAD proteins, with varying paralog selectivity profiles. OPN-9840 is an oral non-covalent pan-TEAD inhibitor displaying dose-dependent and on-target in vitro and in vivo efficacy for NCI-H226, an NF2-mutant MM cell line. OPN-9840 showed equivalent binding in thermal shift assays (10°C delta Tm) for depalmitoylated TEAD1-4 proteins, and blocked TEAD reporter activity (IC50 100nM) and NCI-H226 cell growth (IC50 400nM). In an NCI-H226 xenograft model, OPN-9840 dosed orally for 14 days exhibited dose-dependent tumor growth inhibition (TGI) from 5 to 50 mg/kg PO (88 to >100% TGI) with tumor regression in the 15 (2/8 mice) and 50 mg/kg (4/8 mice) dose groups. qPCR of OPN-9840-treated tumors confirmed 50-70% downregulation of canonical TEAD targets, CTGF and CYR61. OPN-9840 shows a favorable safety profile, including a lack of cytotoxicity (IC50>50µM) in HEK293T and HepG2 cells. Additionally, OPN-9840 caused no body weight loss in the NCI-H226 xenograft model following 14 days of dosing. In vitro evaluation of OPN-9840 in the blood-brain barrier (BBB) specific parallel artificial membrane permeability assay exhibited high BBB penetration potential (-Log Pe = 4.7). High oral exposure was achieved at all dose levels (AUC0-24 > 100,000 h•ng/mL) after 14 days in the NCI-H226 study. We next investigated synergy between TEAD inhibition and targeted therapy in MM. Combined treatment of one of our pan-TEAD covalent compounds, OPN-9652 (from a separate chemical series to OPN-9840), at 50 mg/kg with 1 mg/kg trametinib resulted in NCI-H226 xenograft tumor regression (130% TGI) whereas trametinib alone only led to decreased tumor growth (74% TGI). RNA-Seq of treated tumors revealed that OPN-9652 and trametinib synergistically inhibited TEAD downstream target genes, including ANKRD1 and CTGF. OPN-9652 as a single agent caused significant downregulation of E2F targets, such as MYC and TP53, suggesting decreased proliferation. In summary, OPN-9840 is a potent pan-TEAD inhibitor that shows monotherapy efficacy in MM. As we observed synergy between another pan-TEAD inhibitor, OPN-9652, with trametinib, we are exploring synergy of OPN-9840 with additional targeted therapies in tumor types beyond MM. Citation Format: Pan-Yu Chen, Bernice Matusow, James Tsai, Peipei Li, Hoa Nguyen, Gaston Habets, Gideon Bollag, Somenath Chowdhury. OPN-9840, a non-covalent potent pan-TEAD inhibitor, exhibits single agent efficacy in preclinical malignant mesothelioma models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7264.

Eliminating oncogenic RAS: back to the future at the drawing board.

RAS drug development has made enormous strides in the past ten years, with the first direct KRAS inhibitor being approved in 2021. However, despite the clinical success of covalent KRAS-G12C inhibitors, we are immediately confronted with resistances as commonly found with targeted drugs. Previously believed to be undruggable due to its lack of obvious druggable pockets, a couple of new approaches to hit this much feared oncogene have now been carved out. We here concisely review these approaches to directly target four druggable sites of RAS from various angles. Our analysis focuses on the lessons learnt during the development of allele-specific covalent and non-covalent RAS inhibitors, the potential of macromolecular binders to facilitate the discovery and validation of targetable sites on RAS and finally an outlook on a future that may engage more small molecule binders to become drugs. We foresee that the latter could happen mainly in two ways: First, non-covalent small molecule inhibitors may be derived from the development of covalent binders. Second, reversible small molecule binders could be utilized for novel targeting modalities, such as degraders of RAS. Provided that degraders eliminate RAS by recruiting differentially expressed E3-ligases, this approach could enable unprecedented tissue- or developmental stage-specific destruction of RAS with potential advantages for on-target toxicity. We conclude that novel creative ideas continue to be important to exterminate RAS in cancer and other RAS pathway-driven diseases, such as RASopathies.

Advances in research on 3C-like protease (3CLpro) inhibitors against SARS-CoV-2 since 2020.

COVID-19 caused by SARS-CoV-2 in late 2019 is still threatening global human health. Although some vaccines and drugs are available in the market, controlling the spread of the SARS-CoV-2 virus remains a huge challenge. 3C-like protease (3CLpro) is a highly conserved key protease for SARS-CoV-2 replication, and no relevant homologous protein with a similar cleavage site to 3CLpro has been identified in humans, highlighting that development of 3CLpro inhibitors exhibits great promise for treatment of COVID-19. In this review, the authors describe the structure and function of 3CLpro. To better understand the characteristics of SARS-CoV-2 3CLpro inhibitors, the SARS-CoV-2 3CLpro inhibitors reported since 2020 are classified into peptidomimetic covalent inhibitors, non-peptidomimetic covalent inhibitors and non-covalent small molecule inhibitors, and the representative inhibitors, their biological activities and binding models are highlighted. Collectively, we hope that all the information presented here will provide new insights into the design and development of more effective 3CLpro inhibitors against SARS-CoV-2 as novel anti-coronavirus drugs.

Proteome-Wide Profiling of the Covalent-Druggable Cysteines with a Structure-Based Deep Graph Learning Network.

Covalent ligands have attracted increasing attention due to their unique advantages, such as long residence time, high selectivity, and strong binding affinity. They also show promise for targets where previous efforts to identify noncovalent small molecule inhibitors have failed. However, our limited knowledge of covalent binding sites has hindered the discovery of novel ligands. Therefore, developing in silico methods to identify covalent binding sites is highly desirable. Here, we propose DeepCoSI, the first structure-based deep graph learning model to identify ligandable covalent sites in the protein. By integrating the characterization of the binding pocket and the interactions between each cysteine and the surrounding environment, DeepCoSI achieves state-of-the-art predictive performances. The validation on two external test sets which mimic the real application scenarios shows that DeepCoSI has strong ability to distinguish ligandable sites from the others. Finally, we profiled the entire set of protein structures in the RCSB Protein Data Bank (PDB) with DeepCoSI to evaluate the ligandability of each cysteine for covalent ligand design, and made the predicted data publicly available on website.

Recent Advances in the Discovery of Potent Proteases Inhibitors Targeting the SARS Coronaviruses.

Across the globe, countries are being challenged by the SARS-CoV-2 (COVID-19) pandemic in ways they have never been before. The global outbreak of SARS-CoV-2 with an uncertain fatality rate has imposed extreme challenges on global health. The World Health Organization (WHO) has officially declared the outbreak of COVID-19 a pandemic, after the disease caused by the new coronavirus spread to more than 100 countries. To date, various therapeutic approaches have been proposed and are being implemented to combat this pandemic, but unfortunately, no sovereign remedy has been established yet. Protease enzymes are important targets to develop therapies for the treatment of infections caused by SARS coronaviruses. In this review, an overview is given on recent advances in the discovery of potent protease inhibitors targeting the SARS coronaviruses. Different classes of natural product inhibitors targeting protease enzymes of SARS coronaviruses have been studied in detail along with their structure-activity relationship analysis. This study emphasized important covalent and non-covalent small molecule inhibitors, which effectively inhibited chymotrypsin- like cysteine protease (3CLpro) and papain-like protease (PLpro) of two SARS coronaviruses, i.e., SARS-CoV-1 and SARS-CoV-2. Repurposing of drugs has also been outlined in this study to understand their roles as quick-to-be-identified therapy to combat these zoonotic coronaviruses.

Ensemble Docking Coupled to Linear Interaction Energy Calculations for Identification of Coronavirus Main Protease (3CLpro) Non-Covalent Small-Molecule Inhibitors.

SARS-CoV-2, or severe acute respiratory syndrome coronavirus 2, represents a new strain of Coronaviridae. In the closing 2019 to early 2020 months, the virus caused a global pandemic of COVID-19 disease. We performed a virtual screening study in order to identify potential inhibitors of the SARS-CoV-2 main viral protease (3CLpro or Mpro). For this purpose, we developed a novel approach using ensemble docking high-throughput virtual screening directly coupled with subsequent Linear Interaction Energy (LIE) calculations to maximize the conformational space sampling and to assess the binding affinity of identified inhibitors. A large database of small commercial compounds was prepared, and top-scoring hits were identified with two compounds singled out, namely 1-[(R)-2-(1,3-benzimidazol-2-yl)-1-pyrrolidinyl]-2-(4-methyl-1,4-diazepan-1-yl)-1-ethanone and [({(S)-1-[(1H-indol-2-yl)methyl]-3-pyrrolidinyl}methyl)amino](5-methyl-2H-pyrazol-3-yl)formaldehyde. Moreover, we obtained a favorable binding free energy of the identified compounds, and using contact analysis we confirmed their stable binding modes in the 3CLpro active site. These compounds will facilitate further 3CLpro inhibitor design.

Abstract C049: Identification of novel K-Ras G12C inhibitors using a DNA encoded library platform

Abstract K-Ras is the most frequently mutated oncogene in cancer. Most mutations cluster in codons 12, 13 and 61, and are involved in the development and progression of a wide range of cancers. Drugging K-Ras has been challenging using traditional drug discovery strategies. Covalent inhibition is a viable drug discovery strategy for drugging difficult targets where identification of non-covalent small molecule inhibitors has been challenging. Marketed covalent inhibitors have been identified for various targets including EGFR and BTK. The K-Ras G12C mutation accounts for 12% of all observed K-Ras mutations in cancer and is most prevalent in non-small lung (NSCLC), pancreatic and colorectal cancers. It has recently been shown that this mutated cysteine can be harnessed for the development of specific covalent inhibitors. We performed covalent based-selections for K-Ras G12C utilizing a 1011 member covalent DNA-encoded library. These selections led to the identification of three novel and chemically distinct series of K-Ras G12C-GDP specific covalent inhibitors. Direct target engagement was demonstrated using FAM-labeled probes in cells over-expressing G12C K-Ras. No target engagement was observed with wild-type or other mutants of K-Ras indicating that these compounds are selective for G12C. Co-crystal structures for all 3 series have been obtained validating the covalent linkage to G12C and also demonstrating “series specific” binding modes for all 3 series of inhibitors. Hit to lead efforts have led us to a tool compound, X-498, which is potent and mutant selective inhibitor of K-Ras G12C. X-498 has a biochemical potency <10 nM (2h) in an HTRF based assay and demonstrates a dose dependent inhibition of phospho-ERK (pERK) in the NCI-H358 (G12C) NSCLC cell line after 4h of compound treatment. No effect on pERK is seen in a G12S containing NSCLC (A549) cell line. In vitro cellular viability assessment of X-498 in K-Ras G12C and non-G12C cancer cell lines demonstrate mutant specific killing of cell lines harboring a K-Ras G12C mutation with GI50 <50 nM and a >400-fold selectivity over a wild-type K-Ras cell line. Structure based drug design is currently being used to optimize chemical properties and potency. Assessment of ADME and PK is ongoing. In summary, we have identified novel chemical series selectively targeting K-Ras G12C mutant that are currently being optimized with the goal of developing treatment for cancer patients harboring the K-Ras G12C mutation. Citation Format: Shilpi Arora, Chris Hupp, Sarah Talcott, Usha Narayanan, Diana Gikunju, Mortiz Von Rechenberg, Michael I Monteiro, John P Giulinger, Kyle Denton, Matt Clark, Andrew Ferguson, Andrew J McRiner, John Cuozzo, Christelle Huguet, Anthony D Keefe. Identification of novel K-Ras G12C inhibitors using a DNA encoded library platform [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C049. doi:10.1158/1535-7163.TARG-19-C049

A high-throughput screen to identify novel small molecule inhibitors of the Werner Syndrome Helicase-Nuclease (WRN).

Werner syndrome (WS), an autosomal recessive genetic disorder, displays accelerated clinical symptoms of aging leading to a mean lifespan less than 50 years. The WS helicase-nuclease (WRN) is involved in many important pathways including DNA replication, recombination and repair. Replicating cells are dependent on helicase activity, leading to the pursuit of human helicases as potential therapeutic targets for cancer treatment. Small molecule inhibitors of DNA helicases can be used to induce synthetic lethality, which attempts to target helicase-dependent compensatory DNA repair pathways in tumor cells that are already genetically deficient in a specific pathway of DNA repair. Alternatively, helicase inhibitors may be useful as tools to study the specialized roles of helicases in replication and DNA repair. In this study, approximately 350,000 small molecules were screened based on their ability to inhibit duplex DNA unwinding by a catalytically active WRN helicase domain fragment in a high-throughput fluorometric assay to discover new non-covalent small molecule inhibitors of the WRN helicase. Select compounds were screened to exclude ones that inhibited DNA unwinding by other helicases in the screen, bound non-specifically to DNA, acted as irreversible inhibitors, or possessed unfavorable chemical properties. Several compounds were tested for their ability to impair proliferation of cultured tumor cells. We observed that two of the newly identified WRN helicase inhibitors inhibited proliferation of cancer cells in a lineage-dependent manner. These studies represent the first high-throughput screen for WRN helicase inhibitors and the results have implications for anti-cancer strategies targeting WRN in different cancer cells and genetic backgrounds.

Abstract 2994: Discovery of selective, noncovalent small molecule inhibitors of DNMT1 as an alternative to traditional DNA hypomethylating agents

Abstract Aberrant DNA hypermethylation within promoter regions and subsequent gene silencing are near universal hallmarks of human cancer. Reversal of DNA methylation by a hypomethylating agent, such as decitabine (Dacogen) or azacytidine (Vidaza), has shown clinical benefit for the treatment of heme malignancies. However, these agents have several limitations that preclude their full potential from being realized such as the requirement for IV administration, poor PK properties and a mechanism that requires incorporation into replicating DNA. This indirect, irreversible inhibition of the DNA methyltransferase (DNMT) family (DNMT1, 3a and 3b) and subsequent DNA damage induces significant dose-limiting toxicity thus preventing sufficient target engagement required for maximal demethylation. A drug discovery effort focused on DNMT1, the key family member responsible for maintaining the DNA methylation pattern, was initiated based on the compelling nature of the target coupled with the need for improved agents. A high-throughput screen identified a single dicyanopyridine (DCP) series of reversible, non-DNA incorporating, highly selective inhibitors for DNMT1 over DNMT3a or DNMT3b. Ensuing structure-activity relationship (SAR) optimization of the series led to the discovery of potent tool compounds that in cancer cells induced robust decreases in global DNA methylation, transcriptional activation of many silenced genes, and inhibition of cancer cell growth. In contrast to decitabine where most cells showed a cytotoxic response, our DNMT1 tool inhibitors primarily elicited a cytostatic response. Furthermore, studies in a mouse tumor model revealed decreased DNA methylation and a dose-dependent (1-45 mg/kg, BID) decrease in tumor growth with regression at the highest doses. In summary, a series of potent, selective DNMT1 inhibitors were discovered and refined delivering tool compounds capable of eliciting changes in DNA methylation, transcriptional activation, and tumor regression at well-tolerated doses. Thus demonstrating that selective, non-covalent inhibitors of DNMT1 may provide benefit over traditional DNA incorporating hypomethylating agents. Citation Format: Melissa B. Pappalardi, Mark Cockerill, Jessica L. Handler, Alexandra Stowell, Kathryn Keenan, Christian S. Sherk, Elisabeth A. Minthorn, Charles F. McHugh, Charlotte Burt, Kristen Wong, David T. Fosbenner, Mehul Patel, Jacques Briand, Helai Mohammad, Lourdes Rueda, Andrew Benowitz, Rab Prinjha, Dirk Heerding, Ryan G. Kruger, Ali Raoof, Allan Jordan, Bryan W. King, Michael T. McCabe. Discovery of selective, noncovalent small molecule inhibitors of DNMT1 as an alternative to traditional DNA hypomethylating agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2994.

Potent and Selective Inhibitors of 8-Oxoguanine DNA Glycosylase.

The activity of DNA repair enzyme 8-oxoguanine DNA glycosylase (OGG1), which excises oxidized base 8-oxoguanine (8-OG) from DNA, is closely linked to mutagenesis, genotoxicity, cancer, and inflammation. To test the roles of OGG1-mediated repair in these pathways, we have undertaken the development of noncovalent small-molecule inhibitors of the enzyme. Screening of a PubChem-annotated library using a recently developed fluorogenic 8-OG excision assay resulted in multiple validated hit structures, including selected lead hit tetrahydroquinoline 1 (IC50 = 1.7 μM). Optimization of the tetrahydroquinoline scaffold over five regions of the structure ultimately yielded amidobiphenyl compound 41 (SU0268; IC50 = 0.059 μM). SU0268 was confirmed by surface plasmon resonance studies to bind the enzyme both in the absence and in the presence of DNA. The compound SU0268 was shown to be selective for inhibiting OGG1 over multiple repair enzymes, including other base excision repair enzymes, and displayed no toxicity in two human cell lines at 10 μM. Finally, experiments confirm the ability of SU0268 to inhibit OGG1 in HeLa cells, resulting in an increase in accumulation of 8-OG in DNA. The results suggest the compound SU0268 as a potentially useful tool in studies of the role of OGG1 in multiple disease-related pathways.

Bruton’s tyrosine kinase in chronic inflammation: from pathophysiology to therapy

Bruton's tyrosine kinase (Btk) not only plays a role in differentiation and activa- tion of B-cells but is also involved in regulating signaling in myeloid cell populations, mast cells, platelets, and osteoclasts. Btk plays a critical role in B-cell receptor signaling and has been shown to be involved in CD40 ligation, Toll-like receptor triggering, and Fc-receptor signaling as well, suggesting that targeting Btk might be particularly useful in autoimmune diseases characterized by pathologic antibodies, macrophage activation, and myeloid-derived type I interferon responses. Btk knockout and X-linked immunodeficiency mouse material and X-linked agammaglobulinemia patient samples are powerful tools to examine the role of Btk in health and disease. In addition, the recent development of several covalent and noncovalent small-molecule inhibitors specifically targeting Btk can contribute to our understanding of the functional role of Btk in several cell types and disease mechanisms. Specific Btk inhibitors have been used in both in vitro and in vivo models for autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, systemic sclerosis, and Sjogren's syndrome. The current small-molecule Btk inhibitors have demonstrated potent and selective Btk inhibition in preclinical studies, and some inhibitors have propelled the clinical develop- ment of such molecules toward clinical trials in patients with rheumatoid arthritis. Future studies on the effectiveness of Btk inhibition in other autoimmune diseases could further broaden the understanding of the disease mechanisms as well as lead to new targeted therapies.

Approaches to design non-covalent inhibitors for human granzyme B (hGrB).

A structure-based design campaign for non-covalent small molecule inhibitors of human granzyme B was carried out by means of a virtual screening strategy employing three constraints and probe site-mapping with FTMAP to identify ligand "hot spots". In addition, new scaffolds of diverse structures were subsequently explored with ROCS shape-based superposition methods, following by Glide SP docking, induced fit docking and analysis of QikProp molecular properties. Novel classes of moderately active small molecule blockers (≥25 μM IC50 values) from commercially available libraries were identified, and three novel scaffolds have been synthesized by multi-step procedures. Furthermore, we provide an example of a comprehensive structure-based drug discovery approach to non-covalent inhibitors that relies on the X-ray structure of a covalently bound ligand and suggest that the design path may be compromised by alternative and unknown binding poses.

Discovery, synthesis, and structure-based optimization of a series of N-(tert-butyl)-2-(N-arylamido)-2-(pyridin-3-yl) acetamides (ML188) as potent noncovalent small molecule inhibitors of the severe acute respiratory syndrome coronavirus (SARS-CoV) 3CL protease.

A high-throughput screen of the NIH molecular libraries sample collection and subsequent optimization of a lead dipeptide-like series of severe acute respiratory syndrome (SARS) main protease (3CLpro) inhibitors led to the identification of probe compound ML188 (16-(R), (R)-N-(4-(tert-butyl)phenyl)-N-(2-(tert-butylamino)-2-oxo-1-(pyridin-3-yl)ethyl)furan-2-carboxamide, Pubchem CID: 46897844). Unlike the majority of reported coronavirus 3CLpro inhibitors that act via covalent modification of the enzyme, 16-(R) is a noncovalent SARS-CoV 3CLpro inhibitor with moderate MW and good enzyme and antiviral inhibitory activity. A multicomponent Ugi reaction was utilized to rapidly explore structure-activity relationships within S(1'), S(1), and S(2) enzyme binding pockets. The X-ray structure of SARS-CoV 3CLpro bound with 16-(R) was instrumental in guiding subsequent rounds of chemistry optimization. 16-(R) provides an excellent starting point for the further design and refinement of 3CLpro inhibitors that act by a noncovalent mechanism of action.

Structure-based design of novel groups for use in the P1 position of thrombin inhibitor scaffolds. Part 1: Weakly basic azoles

Despite their relatively weak basicity, simple azoles, specifically imidazoles and aminothiazoles, can function as potent surrogates for the more basic amines (e.g., alkyl amines, amidines, guanidines, etc.) which are most often employed as the P1 ligand in the design of noncovalent small molecule inhibitors of thrombin.

Small molecule inhibitors of urokinase-type plasminogen activator

The urokinase-type plasminogen activator (uPA) protein is a multifunctional protein involved in a myriad of biological activities including extracellular matrix degradation and cell invasion. Active uPA is a 411 amino acid protein consisting of 3 domains, each of which confers a particular biological function to the overall protein. The amino terminal domain or growth factor domain (GFD), comprised of amino acid residues 1 – 48, is involved in uPA interaction with its cell surface receptor, urokinase-type plasminogen activator receptor (UPAR). The interaction of uPA with UPAR promotes, in part, cell adhesion, migration and invasion. A second domain is the kringle domain, comprising amino acid residues 49 – 135. Initially thought to bind heparin, the kringle domain has more recently been shown to possess antiangiogenic activity. A third domain comprising amino acid residues 159 – 411, the serine protease domain, is involved in the proteolytic activation of plasminogen to plasmin. The production of plasmin by uPA begins a cascade of events manifested by extracellular matrix degradation. The recent patent literature describes small molecule compounds, which inhibit the interaction of uPA with UPAR, inhibit the proteolytic activity of the uPA serine protease domain and inhibit the interaction of uPA with its natural inhibitor, plasminogen activator inhibitor-1 (PAI-1). Small peptides encompassing residues 19 – 31 of the GFD have been developed which exhibit potent inhibition of the uPA–UPAR interaction and show efficacy in tumour-bearing animal models. Small molecules have been disclosed by Corvas, which are reported to be inhibitors of PAI-1. Finally, two approaches toward the development of inhibitors of the uPA serine protease domain have been described in the recent patent literature. The first approach describes non-covalent peptidederived inhibitors discovered by phage display techniques, which bind in the substrate-binding groove of the uPA active site. An alternative approach describes non-covalent small molecule inhibitors, which bind in the enzyme active site in a slightly different binding mode than the peptide-derived inhibitors. These small molecule non-peptide analogues inhibit the uPA proteolytic activity quite effectively and are reported to possess excellent enzyme selectivity and highly improved oral activity. The clinical utility of small molecule uPA enzyme inhibitor analogues awaits the results of a preliminary clinical evaluation of compounds described by Wilex.