Identification Of Small Molecule Inhibitors Research Articles

Identification of small molecule inhibitors against Lin28/let-7 to suppress tumor progression and its alleviation role in LIN28-dependent glucose metabolism.

The reciprocal suppression of an RNA-binding protein LIN28 (human abnormal cell lineage 28) and miRNA Let-7 (Lethal 7) is considered to have a prime role in hepatocellular carcinoma (HCC). Though targeting this inhibition interaction is effective for therapeutics, it causes other unfavorable effects on glucose metabolism and increased insulin resistance. Hence, this study aims to identify small molecules targeting Lin28/let-7 interaction along with additional potency to improve insulin sensitivity. Of 22,14,996 small molecules screened by high throughput virtual screening, 6 molecules, namely 41354, 1558, 12437, 23837, 15710, and 8319 were able to block the LIN28 interaction with let-7 and increase the insulin sensitivity via interacting with PPARγ (peroxisome proliferator-activated receptors γ). MM-GBSA (Molecular Mechanics-Generalized Born Surface Area) analysis is used to re-score the binding affinity of docked complexes. Upon further analysis, it is also seen that these molecules have superior ADME (Absorption, Distribution, Metabolism, and Excretion) properties and form stable complexes with the targets for a significant period in a biologically simulated environment (Molecular Dynamics simulation) for 100 ns. From our results, we hypothesize that these identified 6 small molecules can be potential candidates for HCC treatment and the glucose metabolic disorder caused by the HCC treatment.

Identification of small molecule inhibitors of penicillin-binding protein 2a of methicillin-resistant Staphylococcus aureus for the therapeutics of bacterial infection.

The penicillin binding protein 2a (PBP2a) is a key enzyme associated with bacterial cell wall synthesis and bacterial infection. Therefore, targeting PBPa2 offers a promising approach for the therapeutics of bacterial resistance and infection. This study presents a comprehensive analysis of alpha-mangostin as a potential inhibitor of PBPa2. Molecular docking simulations revealed a strong binding affinity between alpha-mangostin and PBP2a, with an affinity score of -6.01 kcal/mol. Notably, alpha-mangostin formed a preferential hydrogen bond with THR216 of PBP2a, alongside several other polar and hydrophobic interactions. ADME and Toxicity predictions indicated that alpha-mangostin possesses favourable pharmacokinetic properties, suggesting its potential as a therapeutic agent. PASS analysis further highlighted its broad range of favourable biological properties. SwissTargetPrediction analysis reinforced these findings, indicating alpha-mangostin's association with various biological processes. Cell toxicity assays demonstrated that alpha-mangostin had no significant impact on the viability of HEK-293 cells, suggesting its potential safety for further development. The IC50 value for alpha-mangostin was found to be 33.43µM. Fluorescence-based binding assays showed that alpha-mangostin effectively inhibited PBP2a activity in a concentration-dependent manner, supporting its role as an inhibitor. In conclusion, the results suggest alpha-mangostin as a promising candidate for inhibiting PBP2a. Further, extensive studies are warranted to explore its clinical applications.

Abstract 5912: Rational design of a small-molecule inhibitor targeting the allosteric site of sos1 in oncogenic k-ras pancreatic cancer

Abstract Oncogenic Kras signaling is a key therapeutic target in human cancers. Ras activation is catalyzed by guanine nucleotide exchange factors (GEFs), of which SOS1 is a major player that transduces receptor tyrosine kinase signaling to Ras. In addition to recently devised oncogenic Kras targeting small molecule inhibitors, disrupting the catalytic interaction between Ras and SOS1 has emerged as an attractive therapeutic strategy in working synergistically with Kras inhibitors and overcoming resistance. Previous studies have demonstrated an active “feed forward” loop of active Kras binding to the allosteric site of SOS1 that further stimulates SOS1 catalytic activation of wild type H/N/Kras, which is required for oncogenic Kras driven tumorigenesis. Here we have developed a rational approach that combines virtual screening with experimental screening to identify small molecule inhibitors that target the allosteric site of SOS1 to suppress oncogenic Kras mediated SOS1-regulated wild-type Ras activities. Unlike SOS1 catalytic site inhibitors which can have significant toxicity due to their ability to inhibit normal Ras signaling function, the lead SOS1 allosteric inhibitors developed in our laboratory binds to the allosteric site of SOS1 and blocks active Ras interaction with SOS1: we found these inhibitors can competitively suppresses SOS1-Ras interaction at the SOS1 allosteric site and dose-dependently inhibits the feed-forward SOS1 GEF activity. Mutagenesis and structure-activity relationship studies mapped the site of action to the SOS1 allosteric site, and preliminarily defined the chemical moieties in the inhibitor essential for the activity. The inhibitor showed dose-dependent and selective efficacy in inhibiting WT Ras activity, downstream signaling activities, and associated cell proliferation in KRAS mutation cells but not KRAS wild-type cells. Compared to the SOS1 catalytic inhibitor, BI3406, our inhibitors showed better selectivity for KRAS mutant pancreatic cancer cells vs. WT KRAS cells. These studies establish a proof of principle for the rational design of small molecule inhibitors targeting an oncogenic Kras specific allosteric site of SOS1 and provide a novel therapeutic concept to target KRAS-driven tumors more effectively. Citation Format: Xin Duan, Chris R Evelyn, Jacek Biesiada, James F Johnson, William L. Seibel, Jaroslaw Meller, Yi Zheng. Rational design of a small-molecule inhibitor targeting the allosteric site of sos1 in oncogenic k-ras pancreatic cancer [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 5912.

Computational analysis of PD-L1 dimerization mechanism induced by small molecules and potential dynamical properties

The design of small molecule inhibitors that target the programmed death ligand-1 (PD-L1) is a forefront issue in immune checkpoint blocking therapy. Small-molecule inhibitors have been shown to exert therapeutic effects by inducing dimerization of the PD-L1 protein, however, the specific mechanisms underlying this dimerization process remain largely unexplored. Furthermore, there is a notable lack of comparative studies examining the binding modes of structurally diverse inhibitors. In view of the research gaps, this work employed molecular dynamics simulations to meticulously examine the interactions between two distinct types of inhibitors and PD-L1 in both monomeric and dimeric forms, and predicted the dimerization mechanism. The results revealed that inhibitors initially bind to a PD-L1 monomer, subsequently attracting another monomer to form a dimer. Notably, symmetric inhibitors observed superior binding efficiency compared to other inhibitors. Key residues, including Ile54, Tyr56, Met115 and Tyr123 played a leading role in binding. Structurally, symmetric inhibitors were capable of thoroughly engaging the binding pocket, promoting a more symmetrical formation of PD-L1 dimers. Furthermore, symmetric inhibitors formed more extensive hydrophobic interactions with protein residues. The insights garnered from this research are expected to significantly contribute to the rational design and optimization of small molecule inhibitors targeting PD-L1.

Recent Advances in Enzyme Inhibition: A Pharmacological Review

Abstract: Enzyme inhibition is a crucial mechanism for regulating biological processes and developing therapeutic interventions. This pharmacological review summarizes recent advances in enzyme inhibition, focusing on key developments and their implications for drug discovery and therapeutic strategies. It explains basic ideas, including the different kinds of inhibitors and how they work, and looks at recent advances in small-molecule inhibitor design, fragment-based drug discovery, and virtual screening techniques. The review also highlights the advances in targeting specific enzyme families, explaining the structural basis of enzyme-inhibitor interactions, optimizing inhibitor potency, selectivity, and pharmacokinetic properties, and new trends in enzyme inhibition. The clinical implications of recent advances in enzyme inhibition include the development of novel therapeutic agents for diseases like cancer, infectious diseases, and neurological disorders. The review addresses challenges and future directions in the field, such as optimizing drug safety, resistance mechanisms, and personalized medicine approaches. Overall, the insights provided in this review may inspire further research and collaborations to accelerate the translation of enzyme inhibitors into effective clinical treatments.

Structure-based design of small molecule inhibitors of the cagT4SS ATPase Cagα of Helicobacter pylori.

We here describe the structure-based design of small molecule inhibitors of the type IV secretion system of Helicobacter pylori. The secretion system is encoded by the cag pathogenicity island, and we chose Cagα, a hexameric ATPase and member of the family of VirB11-like proteins, as target for inhibitor design. We first solved the crystal structure of Cagα in a complex with the previously identified small molecule inhibitor 1G2. The molecule binds at the interface between two Cagα subunits and mutagenesis of the binding site identified Cagα residues F39 and R73 as critical for 1G2 binding. Based on the inhibitor binding site we synthesized 98 small molecule derivates of 1G2 to improve binding of the inhibitor. We used the production of interleukin-8 of gastric cancer cells during H. pylori infection to screen the potency of inhibitors and we identified five molecules (1G2_1313, 1G2_1338, 1G2_2886, 1G2_2889, and 1G2_2902) that have similar or higher potency than 1G2. Differential scanning fluorimetry suggested that these five molecules bind Cagα, and enzyme assays demonstrated that some are more potent ATPase inhibitors than 1G2. Finally, scanning electron microscopy revealed that 1G2 and its derivatives inhibit the assembly of T4SS-determined extracellular pili suggesting a mechanism for their anti-virulence effect.

In silico Analysis of Structure and Function of Hypothetical Proteins in Salmonella typhimurium (SL1344)

Salmonella typhimurium strain SL1344(S.typhi) is a non typhoidal bacteria which may cause infection in human gastrointestinal tract, may cause abdominal pain, fever, diarrhea, and inflammation some times which may lead to the human death. A comparative study of hypothetical outer membrane proteins of Salmonella typhimurium strain SL1344 (S.typhi) was performed using bioinformatic tools. Bioinformatics is a source to analyse whole genomic sequence of a vast number of uncharacterized genes. Online databases like UniProt, String, Pfam and other tools were used. Thirteen uncharacterized and hypothetical protein present in outer membrane of the bacteria S.typhi were retrieved from UniProt and its structure and function were studied. In this study the cellular function, pathway of enzyme interaction, antibiotic resistance were analyzed. The structure of the protein as well the function of these proteins provides better understanding to perform molecular docking and identification of small molecule inhibitors.

Lowering mutant huntingtin by small molecules relieves Huntington’s disease symptoms and progression

Huntington’s disease (HD) is an incurable inherited disorder caused by a repeated expansion of glutamines in the huntingtin gene (Htt). The mutant protein causes neuronal degeneration leading to severe motor and psychological symptoms. Selective downregulation of the mutant Htt gene expression is considered the most promising therapeutic approach for HD. We report the identification of small molecule inhibitors of Spt5-Pol II, SPI-24 and SPI-77, which selectively lower mutant Htt mRNA and protein levels in HD cells. In the BACHD mouse model, their direct delivery to the striatum diminished mutant Htt levels, ameliorated mitochondrial dysfunction, restored BDNF expression, and improved motor and anxiety-like phenotypes. Pharmacokinetic studies revealed that these SPIs pass the blood-brain-barrier. Prolonged subcutaneous injection or oral administration to early-stage mice significantly delayed disease deterioration. SPI-24 long-term treatment had no side effects or global changes in gene expression. Thus, lowering mutant Htt levels by small molecules can be an effective therapeutic strategy for HD.

The identification of small molecule inhibitors with anthelmintic activities that target conserved proteins among ruminant gastrointestinal nematodes.

Gastrointestinal nematode (GIN) infections are a major concern for the ruminant industry worldwide and result in significant production losses. Naturally occurring polyparasitism and increasing drug resistance that potentiate disease outcomes are observed among the most prevalent GINs of veterinary importance. Within the five major taxonomic clades, clade Va represents a group of GINs that predominantly affect the abomasum or small intestine of ruminants. However, the development of effective broad-spectrum anthelmintics against ruminant clade Va GINs has been challenged by a lack of comprehensive druggable genome resources. Here, we first assembled draft genomes for three clade Va species (Cooperia oncophora, Trichostrongylus colubriformis, and Ostertagia ostertagi) and compared them with closely related ruminant GINs. Genome-wide phylogenetic reconstruction showed a relationship among ruminant GINs structured by taxonomic classification. Orthogroup (OG) inference and functional enrichment analyses identified 220 clade Va-specific and Va-conserved OGs, enriched for functions related to cell cycle and cellular senescence. Further transcriptomic analysis identified 61 taxonomically and functionally conserved clade Va OGs that may function as drug targets for new broad-spectrum anthelmintics. Chemogenomic screening identified 11 compounds targeting homologs of these OGs, thus having potential anthelmintic activity. In in vitro phenotypic assays, three kinase inhibitors (digitoxigenin, K-252a, and staurosporine) exhibited broad-spectrum anthelmintic activities against clade Va GINs by obstructing the motility of exsheathed L3 (xL3) or molting of xL3 to L4. These results demonstrate valuable applications of the new ruminant GIN genomes in gaining better insights into their life cycles and offer a contemporary approach to discovering the next generation of anthelmintics.IMPORTANCEGastrointestinal nematode (GIN) infections in ruminants are caused by parasites that inhibit normal function in the digestive tract of cattle, sheep, and goats, thereby causing morbidity and mortality. Coinfection and increasing drug resistance to current therapeutic agents will continue to worsen disease outcomes and impose significant production losses on domestic livestock producers worldwide. In combination with ongoing therapeutic efforts, advancing the discovery of new drugs with novel modes of action is critical for better controlling GIN infections. The significance of this study is in assembling and characterizing new GIN genomes of Cooperia oncophora, Ostertagia ostertagi, and Trichostrongylus colubriformis for facilitating a multi-omics approach to identify novel, biologically conserved drug targets for five major GINs of veterinary importance. With this information, we were then able to demonstrate the potential of commercially available compounds as new anthelmintics.

Identification of small molecule inhibitors of G3BP-driven stress granule formation.

Stress granule formation is triggered by the release of mRNAs from polysomes and is promoted by the action of the RNA-binding proteins G3BP1/2. Stress granules have been implicated in several disease states, including cancer and neurodegeneration. Consequently, compounds that limit stress granule formation or promote their dissolution have potential as both experimental tools and novel therapeutics. Herein, we describe two small molecules, G3BP inhibitor a and b (G3Ia and G3Ib), designed to bind to a specific pocket in G3BP1/2 that is targeted by viral inhibitors of G3BP1/2 function. In addition to disrupting the co-condensation of RNA, G3BP1, and caprin 1 in vitro, these compounds inhibit stress granule formation in cells treated prior to or concurrent with stress and dissolve pre-existing stress granules. These effects are consistent across multiple cell types and a variety of initiating stressors. Thus, these compounds represent powerful tools to probe the biology of stress granules and hold promise for therapeutic interventions designed to modulate stress granule formation.

Targeting Allosteric Site of PCSK9 Enzyme for the Identification of Small Molecule Inhibitors: An In Silico Drug Repurposing Study.

The primary cause of atherosclerotic cardiovascular disease (ASCVD) is elevated levels of low-density lipoprotein cholesterol (LDL-C). Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a crucial role in this process by binding to the LDL receptor (LDL-R) domain, leading to reduced influx of LDL-C and decreased LDL-R cell surface presentation on hepatocytes, resulting higher circulating levels of LDL-C. As a consequence, PCSK9 has been identified as a crucial target for drug development against dyslipidemia and hypercholesterolemia, aiming to lower plasma LDL-C levels. This research endeavors to identify promising inhibitory candidates that target the allosteric site of PCSK9 through an in silico approach. To start with, the FDA-approved Drug Library from Selleckchem was selected and virtually screened by docking studies using Glide extra-precision (XP) docking mode and Smina software (Version 1.1.2). Subsequently, rescoring of 100 drug compounds showing good average docking scores were performed using Gnina software (Version 1.0) to generate CNN Score and CNN binding affinity. Among the drug compounds, amikacin, bestatin, and natamycin were found to exhibit higher docking scores and CNN affinities against the PCSK9 enzyme. Molecular dynamics simulations further confirmed that these drug molecules established the stable protein-ligand complexes when compared to the apo structure of PCSK9 and the complex with the co-crystallized ligand structure. Moreover, the MM-GBSA calculations revealed binding free energy values ranging from -84.22 to -76.39 kcal/mol, which were found comparable to those obtained for the co-crystallized ligand structure. In conclusion, these identified drug molecules have the potential to serve as inhibitors PCSK9 enzyme and these finding could pave the way for the development of new PCSK9 inhibitory drugs in future in vitro research.

Solubilizer Tag Effect on PD-L1/Inhibitor Binding Properties for m-Terphenyl Derivatives.

Although heavily studied, the subject of anti-PD-L1 small-molecule inhibitors is still elusive. Here we present a systematic overview of the principles behind successful anti-PD-L1 small-molecule inhibitor design on the example of the m-terphenyl scaffold, with a particular focus on the neglected influence of the solubilizer tag on the overall affinity toward PD-L1. The inhibitor developed according to the proposed guidelines was characterized through its potency in blocking PD-1/PD-L1 complex formation in homogeneous time-resolved fluorescence and cell-based assays. The affinity is also explained based on the crystal structure of the inhibitor itself and its costructure with PD-L1 as well as a molecular modeling study. Our results structuralize the knowledge related to the strong pharmacophore feature of the m-terphenyl scaffold preferential geometry and the more complex role of the solubilizer tag in PD-L1 homodimer stabilization.

Uncovering the non-histone interactome of the BRPF1 bromodomain using site-specific azide-acetyllysine photochemistry

Bromodomain-PHD finger protein 1 (BRPF1) belongs to the BRPF family of bromodomain-containing proteins. Bromodomains are exclusive reader modules that recognize and bind acetylated histones and non-histone transcription factors to regulate gene expression. The biological functions of acetylated histone recognition by BRPF1 bromodomain are well characterized; however, the function of BRPF1 regulation via non-histone acetylation is still unexplored. Therefore, identifying the non-histone interactome of BRPF1 is pivotal in deciphering its role in diverse cellular processes, including its misregulation in diseases like cancer. Herein, we identified the non-histone interacting partners of BRPF1 utilizing a protein engineering-based approach. We site-specifically introduced the unnatural photo-cross-linkable amino acid 4-azido-L-phenylalanine (AzF) into the bromodomain of BRPF1 without altering its ability to recognize acetylated histone proteins. Upon photo-irradiation, the engineered BRPF1 generates a reactive nitrene species, cross-linking interacting partners with spatio-temporal precision. We demonstrated the robust cross-linking efficiency of the engineered variant with reported histone ligands of BRPF1 and further used the variant reader to cross-link its interactome. We also characterized novel interacting partners by proteomics, suggesting roles for BRPF1 in diverse cellular processes. BRPF1 interaction with ILF3, one of these novel interacting partners, was further validated by ITC and co-IP. Lastly, we used publicly available ChIP-seq and RNA-seq datasets to understand the colocalization of BRPF1 and ILF3 in regulating gene expression in the context of hepatocellular carcinoma. Together, these results will be crucial for full understanding of the roles of BRPF1 in transcriptional regulation and in the design of small-molecule inhibitors for cancer treatment.

Abstract A170: Discovery of small molecule inhibitors of ADAR1

Abstract Background: Adenosine deaminase acting on RNA 1 (ADAR1) is an RNA editing enzyme that catalyzes the deamination of adenosine to inosine in double-stranded (ds) RNA. Cellular dsRNAs are edited by ADAR1 to prevent their recognition by cytoplasmic dsRNA sensors such as MDA5 and PKR. Loss of ADAR1 has been shown to induce the activation of these sensors in cancer cells with high intrinsic type 1 interferon signaling, leading to cell death. Accent has developed a Type I Interferon Stimulated Gene (TISG) score through analysis of publicly available datasets that is predictive of sensitivity to ADAR1 loss. As 15-30% of primary tumors in TCGA display elevated type I interferon signaling, ADAR1 has become an attractive oncology target. Here we describe the development of an in vitro and cellular assay suite that was used to progress chemical matter identified from a high-throughput screen (HTS) into single-digit nanomolar inhibitors of cellular ADAR1. Materials and Methods: A HAP-1 cell model of ADAR1 loss was used in rescue experiments to validate a small molecule inhibition approach towards the targeting of ADAR1 activity. A high-throughput screening-compatible biochemical assay was developed and applied to screen diversity libraries, leading to the identification of a chemical series of ADAR1 inhibitors. The mechanism of inhibition of the series was characterized by substrate-competition and orthogonal substrate binding assays. Iterative optimization of the series resulted in cell-permeable compounds of sufficient potency to be tested in a suite of ADAR1 cellular assays. Results: ADAR1 loss or catalytic site-mutant cells were more sensitive to exogenous Interferon-β than parental cells, leading to decreased growth and viability of HAP-1 cells. We demonstrate knock-out of ADAR1 or loss of catalytic activity results in activation of PKR upon interferon-β treatment in the HAP-1 model, unlike parental cells. Development of a label-free biochemical assay of sufficient sensitivity to be run under steady-state conditions, showing linear reaction progress curves over multiple rounds of ADAR1 turnover, enabled high-throughput screening. A hit series was identified that showed RNA substrate non-competitive behavior in orthogonal RNA binding assays. To further assist understanding of series mechanism, a crystallization system was developed that produced a novel human ADAR1 structure to 1.45Å resolution. A suite of in vitro and cellular assays were used to further optimize and identify small molecules with robust inhibition of cellular ADAR1, as read out by cellular RNA editing assays, as well as activation of downstream dsRNA sensor pathways: secretion of interferon B and phosphorylation of PKR. Consistent with our target hypothesis, treatment of the TISG-high squamous cell carcinoma OE21 cell line with potent ADAR1 inhibitors results in cell growth inhibition. Conclusions: Accent developed a suite of assays that led to the identification of small molecule inhibitors targeting ADAR1. Citation Format: Shane M Buker, Stephen J Blakemore, P. Ann Boriack-Sjodin, Cindy Collins, Robert A Copeland, Kenneth W Duncan, Anna Ericsson, Alexandra Garadino, April Greene-Colozzi, Nathalie Leger, Gordon Lockbaum, Anugraha Raman, Scott Ribich, Allen Sickmier, Brian S Sparling, Jie Wu, Serena Silver. Discovery of small molecule inhibitors of ADAR1 [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A170.

Abstract A001: Mapping the interaction between C-type lectin domain group 14A and Multimerin 2

Abstract Introduction: Understanding the molecular basis of protein-protein interactions involved in the regulation of angiogenesis enhances our understanding of cancer pathogenesis and enables the development of novel anti-cancer therapies. In this study, we focused on the transmembrane spanning glycoprotein C-type lectin domain group 14A (CLEC14A), which is exclusively expressed on the tumor endothelium and is important in regulating tumor growth. CLEC14A promotes endothelial tube formation and migration in vitro, in addition tumor growth is retarded in CLEC14A knockout mice. CLEC14A, through its N-terminal C-type lectin domain (CTLD) interacts with the extracellular matrix protein Multimerin 2 (MMRN2) and heparan sulfate proteoglycans. Administration of antibodies which block the CLEC14A-MMRN2 interaction decreases tumor growth in vivo indicating the importance of this interaction, however the biological significance of CLEC14A’s binding to heparan sulfate proteoglycans is not known. Aims: The purpose of this study was to identify the area and key residues on CLEC14A which interact directly with MMRN2. Mapping the CLEC14A-MMRN2 interface would enable the design of small molecule inhibitors of this interaction, a potential anti-cancer strategy. Methods: We employed the Alpha fold predictive modelling algorithm to analyze the predicted structure of CLEC14A. Solvent-facing residues within a previously identified critical loop region were mutated to alanine and binding to MMRN2 was determined. Flow cytometry was used to assess the cell surface expression of these CLEC14A mutants, and far western blotting was employed to determine their capacity to bind MMRN2. Furthermore, we studied whether these mutants interacted with heparin-bound agarose beads in pulldown assays. Results: The alanine-scanning approach resulted in the identification of specific residues of CLEC14A which directly interacted with MMRN2. Furthermore, we identified the epitope on CLEC14A for a biologically active antibody previously reported to block the CLEC14A-MMRN2 interaction. Additionally, we identified critical residues that are essential for cell surface trafficking of CLEC14A. Importantly, the CLEC14A mutants that do not bind to MMRN2 retain their capacity to bind heparin. Conclusion: By leveraging predictive modelling software, we have characterized the molecular interaction between CLEC14A and MMRN2. Our findings may inform anti-angiogenesis drug design in the future. Citation Format: Aleen Baber, Victoria L Birmingham, Roy Bicknell, Philip R Morrison. Mapping the interaction between C-type lectin domain group 14A and Multimerin 2 [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A001.

Identification of Small Molecule Inhibitors and Ligand Directed Degraders of Calcium/Calmodulin Dependent Protein Kinase Kinase 1 and 2 (CaMKK1/2).

CaMKK2 signals through AMPK-dependent and AMPK-independent pathways to trigger cellular outputs including proliferation, differentiation, and migration, resulting in changes to metabolism, bone mass accrual, neuronal function, hematopoiesis, and immunity. CAMKK2 is upregulated in tumors including hepatocellular carcinoma, prostate, breast, and gastric cancer, and genetic deletion in myeloid cells results in increased antitumor immunity in several syngeneic models. Validation of the biological roles of CaMKK2 has relied on genetic deletion or small molecule inhibitors with activity against several biological targets. We sought to generate selective inhibitors and degraders to understand the biological impact of inhibiting catalytic activity and scaffolding and the potential therapeutic benefits of targeting CaMKK2. We report herein selective, ligand-efficient inhibitors and ligand-directed degraders of CaMKK2 that were used to probe immune and tumor intrinsic biology. These molecules provide two distinct strategies for ablating CaMKK2 signaling in vitro and in vivo.

Mutate and Conjugate: A Method to Enable Rapid In-Cell Target Validation.

Target validation remains a challenge in drug discovery, which leads to a high attrition rate in the drug discovery process, particularly in Phase II clinical trials. Consequently, new approaches to enhance target validation are valuable tools to improve the drug discovery process. Here, we report the combination of site-directed mutagenesis and electrophilic fragments to enable the rapid identification of small molecules that selectively inhibit the mutant protein. Using the bromodomain-containing protein BRD4 as an example, we employed a structure-based approach to identify the L94C mutation in the first bromodomain of BRD4 [BRD4(1)] as having a minimal effect on BRD4(1) function. We then screened a focused, KAc mimic-containing fragment set and a diverse fragment library against the mutant and wild-type proteins and identified a series of fragments that showed high selectivity for the mutant protein. These compounds were elaborated to include an alkyne click tag to enable the attachment of a fluorescent dye. These clickable compounds were then assessed in HEK293T cells, transiently expressing BRD4(1)WT or BRD4(1)L94C, to determine their selectivity for BRD4(1)L94C over other possible cellular targets. One compound was identified that shows very high selectivity for BRD4(1)L94C over all other proteins. This work provides a proof-of-concept that the combination of site-directed mutagenesis and electrophilic fragments, in a mutate and conjugate approach, can enable rapid identification of small molecule inhibitors for an appropriately mutated protein of interest. This technology can be used to assess the cellular phenotype of inhibiting the protein of interest, and the electrophilic ligand provides a starting point for noncovalent ligand development.

A systematic pipeline of protein structure selection for computer-aided drug discovery: A case study on T790M/L858R mutant EGFR structures.

Virtual screening (VS) is a routine method to evaluate chemical libraries for lead identification. Therefore, the selection of appropriate protein structures for VS is an essential prerequisite to identify true actives during docking. But the presence of several crystal structures of the same protein makes it difficult to select one or few structures rationally for screening. Therefore, a computational prioritization protocol has been developed for shortlisting crystal structures that identify true active molecules with better efficiency. As identification of small-molecule inhibitors is an important clinical requirement for the T790M/L858R (TMLR) EGFR mutant, it has been selected as a case study. The approach involves cross-docking of 21 co-crystal ligands with all the structures of the same protein to select structures that dock non-native ligands with lower RMSD. The cross docking performance was then correlated with ligand similarity and binding-site conformational similarity. Eventually, structures were shortlisted by integrating cross-docking performance, and ligand and binding-site similarity. Thereafter, binding pose metadynamics was employed to identify structures having stable co-crystal ligands in their respective binding pockets. Finally, different enrichment metrics like BEDROC, RIE, AUAC, and EF1% were evaluated leading to the identification of five TMLR structures (5HCX, 5CAN, 5CAP, 5CAS, and 5CAO). These structures docked a number of non-native ligands with low RMSD, contain structurally dissimilar ligands, have conformationally dissimilar binding sites, harbor stable co-crystal ligands, and also identify true actives early. The present approach can be implemented for shortlisting protein targets of any other important therapeutic kinases.

Abstract IA009: Development of HIF2 inhibitors: Bench to bedside and back

Abstract Arguably the most important driver of clear cell renal cell carcinoma (ccRCC), HIF2a was traditionally regarded as undruggable. However, structural analyses at UT Southwestern Medical Center found a vulnerability leading to the identification of small molecule inhibitors, and eventually the development of highly related PT drugs by Peloton Therapeutics (PT2385 and PT2977, also called belzutifan). PT drugs bind to HIF2a and induce a conformational change that triggers its dissociation from HIF1b, thereby inhibiting DNA binding and gene transactivation. PT drugs have shown activity in patients, but resistance develops over time. Resistance mutations have been identified, initially in preclinical models, and subsequently in patients. This has spurred the development of second-generation inhibitors, including siRNA-based therapeutics. Recently, we reported a tumor-directed HIF2a-targeting siRNA drug (siHIF2) developed by Arrowhead Pharmaceuticals that effectively depleted both wild-type and mutant HIF2a. siHIF2 showed activity against ccRCC in preclinical models and in patients. Overall, these studies establish HIF2a as a core dependency and therapeutic target, and set a paradigm for tumor-directed RNA-based therapeutics in cancer. Citation Format: James Brugarolas. Development of HIF2 inhibitors: Bench to bedside and back [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr IA009.

Computational-based drug design of novel small molecules targeting p53-MDMX interaction

The regulation of the p53 tumor suppressor pathway is critically dependent on the activity of Murine Double Minute 2 (MDM2) and Murine Double Minute X (MDMX) proteins. In certain types of cancer cells, excessive amount of MDMX can poly-ubiquitinate p53, which can result in its degradation, leading to a subsequent reduction in the levels of this protein. Therefore, the design of small-molecule inhibitors targeting the MDMX-p53 interaction has emerged as a promising strategy for cancer therapy. In this study, we employed computational techniques including pharmacophore modeling and molecular docking to identify three potential small molecule inhibitors (CID_25094615, CID_137634453, and CID_25094344) of the MDMX-p53 interaction from a PubChem database. Molecular dynamics of 100000 ps were conducted to assess the stability of the MDMX-inhibitor complexes. Our results showed that all three compounds exhibit stable binding with MDMX, with significantly lower root mean square deviation (RMSD) and fluctuation (RMSF) values than the control ligand, indicating superior stability. Additionally, the three compounds exhibit stronger intermolecular hydrogen bond (HBOND) interactions compared to the control, suggesting stronger stability. Overall, our findings highlight the potential of these compounds as lead candidates for the development of novel anticancer agents that target the MDMX-p53 interaction. Communicated by Ramaswamy H. Sarma