Potent Small Molecule Inhibitor Research Articles

Preclinical and phase 1/2 data of the CHK1 inhibitor BBI-355 in development for esophageal and gastric cancers (EGC) with EGFR or FGFR2 amplifications.

TPS517 Background: High-copy number amplifications of oncogenes (e.g., EGFR , FGFR2 ) frequently occur on extrachromosomal DNA (ecDNA), highly transcribed units of circular non-chromosomal DNA. While targeted therapies have improved survival for patients with oncogene mutations, they have limited activity in oncogene amplified cancers. In Barrett’s esophagus-associated esophageal adenocarcinoma (EAC) ecDNA can be found early in the transition from high-grade dysplasia to EAC. In EAC and gastric cancer (EGC), ~7% and ~3% have EGFR and FGFR2 amplifications, respectively, with possibly >50% occurring on ecDNA. Tumor cells with oncogene amplifications, particularly on ecDNA, have increased DNA replication stress and are sensitive to inactivation of checkpoint kinase 1 (CHK1). We developed BBI-355, an oral, potent, and selective small molecule inhibitor of CHK1, which is in Phase 1/2 clinical development. Methods: BBI-355-101 is a first-in-human Phase 1/2 study of BBI-355 alone or in combination with targeted therapies for advanced or metastatic solid tumors with oncogene amplification (NCT05827614). Part 1 is dose escalation of BBI-355 (PO Q2D). In Parts 2 and 3, patients with EGFR or FGFR 1-3 amplifications are treated with BBI-355 in combination with the EGFR inhibitor erlotinib (150 mg PO QD) or the FGFR inhibitor futibatinib (20 mg PO QD). While this study is enrolling all tumor types, EGC are some of the most likely to harbor ecDNA amplifications. Amplification-driven cancers rarely contain driver oncogene mutations or fusions, and so are excluded. Results: Preclinical: Tumors that harbor oncogene amplifications on ecDNA can evade targeted therapeutic pressure via ecDNA-based resistance mechanisms. BBI-355 in combination with agents targeting the amplified oncogenes ( FGFR2 or EGFR ) prevented resistance to targeted therapies in multiple ecDNA+ gastric cancer CDX and PDX models. Clinical: Safety and PK data of BBI-355 alone and in combination with erlotinib or futibatinib will be presented. The most common drug-related TEAEs, SAEs, and DLTs were hematologic (i.e., leukopenia, neutropenia, lymphopenia and thrombocytopenia) which are on target for CHK1 inhibition and generally well managed. Dose escalation is ongoing and the RP2D/MTD has not been determined. PK showed dose-dependent increase in exposure of BBI-355. Target engagement as determined by pCHK1 IHC as a PD biomarker was observed at all dose levels of BBI-355. Conclusions: The first ecDNA directed therapy, BBI-355, demonstrated significant synergistic anti-tumor activity in combination with targeted therapies in multiple ecDNA+ oncogene amplified EGC models. BBI-355 alone and in combination with erlotinib/futibatinib was well tolerated. Clinical testing in patients with EGFR and FGFR2 oncogene amplifications is ongoing with a strong rational for EGC. Clinical trial information: NCT05827614 .

Metabolic adaptations to acute glucose uptake inhibition converge upon mitochondrial respiration for leukemia cell survival

One hallmark of cancer is the upregulation and dependency on glucose metabolism to fuel macromolecule biosynthesis and rapid proliferation. Despite significant pre-clinical effort to exploit this pathway, additional mechanistic insights are necessary to prioritize the diversity of metabolic adaptations upon acute loss of glucose metabolism. Here, we investigated a potent small molecule inhibitor to Class I glucose transporters, KL-11743, using glycolytic leukemia cell lines and patient-based model systems. Our results reveal that while several metabolic adaptations occur in response to acute glucose uptake inhibition, the most critical is increased mitochondrial oxidative phosphorylation. KL-11743 treatment efficiently blocks the majority of glucose uptake and glycolysis, yet markedly increases mitochondrial respiration via enhanced Complex I function. Compared to partial glucose uptake inhibition, dependency on mitochondrial respiration is less apparent suggesting robust blockage of glucose uptake is essential to create a metabolic vulnerability. When wild-type and oncogenic RAS patient-derived induced pluripotent stem cell acute myeloid leukemia (AML) models were examined, KL-11743 mediated induction of mitochondrial respiration and dependency for survival associated with oncogenic RAS. Furthermore, we examined the therapeutic potential of these observations by treating a cohort of primary AML patient samples with KL-11743 and witnessed similar dependency on mitochondrial respiration for sustained cellular survival. Together, these data highlight conserved adaptations to acute glucose uptake inhibition in diverse leukemic models and AML patient samples, and position mitochondrial respiration as a key determinant of treatment success.

P1002 Changes in circulating lymphocyte subsets and CCR9 transcripts as mechanistic biomarkers of the small molecule α4β7 inhibitor MORF-057 in patients with Ulcerative Colitis

Abstract Background MORF-057 is an orally administered, selective, and potent small molecule inhibitor of the cell surface receptor integrin a4b7. Upon binding to the a4b7 heterodimer, MORF-057 blocks the interaction of this integrin with the endothelial ligand mucosal vascular addressin cell adhesion molecule 1 (MAdCAM-1) which, in turn, prevents the extravasation of circulating lymphocytes into the intestinal mucosa. In this study, the pharmacodynamic effects of MORF-057 on immune cell trafficking were evaluated in UC patients using a flow cytometry-based assay and by assessing the expression of CCR9 gene in blood. Methods As part of the induction phase of an open-label, single-arm, Phase 2a study (NCT05291689) the efficacy, safety, and tolerability of MORF-057 in 35 patients with moderately to severely active UC disease were evaluated. Patients were treated with MORF-057 at a dose of 100 mg BID orally for an induction period of 12 weeks followed by a 40-week maintenance period. Blood was collected for peripheral blood mononuclear cell (PBMC) isolation and RNA extraction at baseline and post-treatment at Week 2, 6, 12, 20, 28, 36, 44, and 52. Using flow cytometry, levels of CD4+β7+ naïve, CD4+β7+ central memory (TCM), CD4+β7 Hi effector memory (TEM) T cells, and CD19+β7+ switched memory B cells (BSM) subpopulations were quantified as a percentage of the parent populations CD4 naive, TCM, TEM and BSM, respectively, then normalized to pre-treatment baseline. Expression of CCR9 mRNA in the blood was quantified as a fold ratio relative to baseline for the same timepoints. Statistical analyses were performed using the Wilcoxon Signed Rank test. Results The percentages of circulating lymphocyte subsets CD4+β7 Hi TEM cells, CD4+β7+ TCM and CD19+β7+ BSM were significantly elevated relative to baseline as early as Week 2 and up to Week 52 (Table 1). These changes were not observed in the overall lymphocyte counts nor in the CD4+β7+Naïve T cells (except at Week 52). The baseline-normalized expression levels of CCR9 at Weeks 12 and 52 were 1.6 [0.6] (mean [SD]) and 1.7 [0.8], respectively (Figure 1). Conclusion Patients receiving MORF-057 for a period of up to 52 weeks achieved statistically significant increases in specific β7+ lymphocyte subsets expressing ITGβ7. Elevated levels of the CCR9 transcript were observed in blood over the course of treatment. Effects observed were consistent with those reported for patients receiving biologic inhibitors of the same pathway as well as in healthy volunteers receiving MORF-0571,2. These changes in circulating lymphocyte populations and CCR9 transcript levels may be used as mechanistic biomarkers for MORF-057 in UC patients.

Computer-aided drug design approaches for the identification of potent inhibitors targeting elongation factor G of Mycobacterium tuberculosis.

Elongation factor G (EF-G) is essential for protein synthesis in Mycobacterium tuberculosis (Mtb), positioning it as a promising target for anti-tubercular drug development. This study employs Structure-Based Drug Design (SBDD) to identify potential small molecule inhibitors that specifically target EF-G. Initially, binding hotspots on EF-G were pinpointed, and the binding modes of various compounds were analyzed. Through protein-protein interaction studies, several promising candidates were validated. Virtual screening and molecular docking techniques were utilized to evaluate the binding affinities and interactions of 20 candidate molecules with Mtb EF-G. Additionally, toxicity profiles of these compounds were assessed using predictive models, which indicated non-carcinogenic properties. To further refine the selection process, Support Vector Machine (SVM) and Random Forest models were applied to predict cell wall permeability. Notably, Asinex (8853) and Asinex (102619) emerged as top candidates, boasting high probability scores for effective permeability. Molecular docking and molecular dynamics (MD) simulations revealed that Asinex (8853), Asinex (102619), and Otava (79226) exhibited strong binding affinities and favorable conformations within the active site of Mtb EF-G. These findings suggest that these compounds have significant potential as inhibitors, warranting further investigation into their efficacy as novel anti-tubercular agents. Overall, this study emphasizes the value of Structure-Based Drug Design in identifying promising therapeutic candidates against tuberculosis by targeting essential bacterial factors like EF-G.

Transforming growth factor beta receptor type I (TGF-βRI) kinase inhibitors IOA-359 and IOA-360 stimulate erythropoiesis in MDS

Overactivation of the Transforming Growth Factor Beta (TGF-β) pathway is implicated in the pathogenesis of cytopenias in Myelodysplastic syndromes (MDS) and Acute Myeloid Leukemia (AML). IOA-359 and IOA-360 are potent small molecule inhibitors of the TGF-beta Receptor type I kinase (TGF-βRI, also referred to as ALK5, activin receptor-like kinase 5) that abrogate SMAD phosphorylation in hematopoietic cell lines. Both inhibitors were able to inhibit TGF-β mediated gene transcription at specific doses. ALK5 inhibitors abrogated the growth inhibitory effects of TGF-β on healthy hematopoietic stem cells and stimulated hematopoietic differentiation in cell lines and MDS/AML specimens. These data demonstrate preclinical efficacy of two novel ALK5 inhibitors, IOA-359 and IOA-360, in stimulating erythroid differentiation in MDS and AML.

Mechanism to disrupt ESCRT-mediated intracellular trafficking through Vps28-small molecules interaction: an in silico approach

The ESCRT (Endosomal Sorting Complex Required for Transport) machinery comprising protein complexes ESCRT-0 to ESCRT-III and Vps4 plays a pivotal role in intracellular trafficking, a process of endocytosing cell surface proteins into the cell for various biological activities. The ESCRT protein complexes are sequentially assembled which interact amongst each other to form a functional ESCRT machinery. Deregulation of these events are shown to be involved in various disease development including tumor formation and viral infections. Recently upregulation of a crucial ESCRT protein, Vps28 has been shown to be implicated in tumor formation. However, Vps28 in ESCRT-I interacts with Vps36 in ESCRT-II to function as a connecting protein during ESCRT machinery formation. Until now biomolecular approaches to inhibit the formation/assembly of ESCRT machinery have not been developed. Hence, we hypothesized that disrupting Vps28/Vps36 interaction would prevent assembly of ESCRT machinery and offer therapeutic potential to restrict disease development and progression. To address this, we utilized a virtual screening approach using a flavonoid-based library to identify potential small molecule inhibitors that can bind to Vps28 active site. Based on the binding affinity, top-hit compounds were identified. Molecular dynamics simulations set over a 500 ns timescale demonstrated the stability of the Vps28-small molecule complexes. Per-residue decomposition analysis using Molecular Mechanics/Poisson–Boltzmann surface area highlighted the significant contributions of active site residues Asn189, Arg190, Arg193 and Asn210 in Vps28 for interaction with small molecules. Absorption, Distribution, Metabolism, Excretion, and Toxicity analysis for toxicity evaluation indicates that molecules Z0131, H0194, Z0199 and DQ00112 exhibited physicochemical properties suitable for drug development.

Molecular Docking and Molecular Dynamics Simulation of New Potential JAK3 Inhibitors.

JAK3 kinase inhibitor has become an effective means to treat tumors and autoimmune diseases. In this study, molecular docking and molecular dynamics simulation were used to study the theoretical interaction mechanism between 1-phenylimidazolidine-2-one molecules and JAK3 protein. The results of molecular docking showed that the six 1-phenylimidazolidine-2-one derivatives obtained by virtual screening were bound to the ATP pocket of JAK3 kinase, which were competitive inhibitors of ATP, and were mainly bound to the pocket through hydrogen bonding and hydrophobic interaction. Further, MM/GBSA based on molecular dynamics simulation sampling was used to calculate the binding energy between six molecules and the JAK3 kinase protein. Subsequently, the binding energy was decomposed into the contribution of each amino acid residue, of which Leu905, Lys855, Asp967, Leu956, Tyr904, and Val836 were the main energycontributing residues. Among them, the molecule numbered LCM01415405 can interact with the specific amino acid Arg911 of JAK3 kinase, suggesting that the molecule may be a selective JAK3 kinase inhibitor. The root-mean-square fluctuation (RMSF) of JAK3 kinase pocket residues during molecular dynamics simulation showed that the combination of six new potential small molecule inhibitors with JAK3 kinase could reduce the flexibility of JAK3 kinase pocket residues. These findings reveal the mechanism of 1-phenylimidazolidine-2-one derivatives on JAK3 protein and provide a relatively solid theoretical basis for the development and structural optimization of JAK3 protein inhibitors.

Metabolic Adaptations To Acute Glucose Uptake Inhibition Converge Upon Mitochondrial Respiration For Leukemia Cell Survival.

One hallmark of cancer is the upregulation and dependency on glucose metabolism to fuel macromolecule biosynthesis and rapid proliferation. Despite significant pre-clinical effort to exploit this pathway, additional mechanistic insights are necessary to prioritize the diversity of metabolic adaptations upon acute loss of glucose metabolism. Here, we investigated a potent small molecule inhibitor to Class I glucose transporters, KL-11743, using glycolytic leukemia cell lines and patient-based model systems. Our results reveal that while several metabolic adaptations occur in response to acute glucose uptake inhibition, the most critical is increased mitochondrial oxidative phosphorylation. KL-11743 treatment efficiently blocks the majority of glucose uptake and glycolysis, yet markedly increases mitochondrial respiration via enhanced Complex I function. Compared to partial glucose uptake inhibition, dependency on mitochondrial respiration is less apparent suggesting robust blockage of glucose uptake is essential to create a metabolic vulnerability. When wild-type and oncogenic RAS patient-derived induced pluripotent stem cell acute myeloid leukemia (AML) models were examined, KL-11743 mediated induction of mitochondrial respiration and dependency for survival associated with oncogenic RAS. Furthermore, we examined the therapeutic potential of these observations by treating a cohort of primary AML patient samples with KL-11743 and witnessed similar dependency on mitochondrial respiration for sustained cellular survival. Together, these data highlight conserved adaptations to acute glucose uptake inhibition in diverse leukemic models and AML patient samples, and position mitochondrial respiration as a key determinant of treatment success.

CPIScore: A Deep Learning Approach for Rapid Scoring and Interpretation of Protein-Ligand Binding Interactions.

Protein-ligand binding affinity prediction is a crucial and challenging task in the field of drug discovery. However, traditional simulation-based computational approaches are often prohibitively time-consuming, limiting their practical utility. In this study, we introduce a novel deep learning method, CPIScore, which leverages the capabilities of Transformer and Graph Convolutional Networks (GCN) to enhance the prediction of protein-ligand binding affinity. CPIScore utilizes the Transformer architecture to capture comprehensive global contexts of protein and ligand sequences, while the GCN component effectively extracts local features from small molecular graphs. Our results demonstrate that CPIScore surpasses both traditional machine learning and other deep learning models in accuracy, achieving a Pearson's r of 0.74 on our test set. Furthermore, CPIScore has been validated across multiple targets, proving its ability to discern inhibitors from a diverse compound library with high enrichment rates. Notably, when applied to a generated focused library of compounds, CPIScore successfully identified six potent small-molecule inhibitors of ATR, which were tested experimentally and four small molecules exhibited inhibitory activity below ten nanomoles. These results highlight CPIScore's potential to significantly streamline and enhance the efficiency of drug discovery processes.

COMPUTATIONAL SCREENING OF POTENT ANTI-INFLAMMATORY COMPOUNDS FOR HUMAN MITOGEN-ACTIVATED PROTEIN KINASE: A COMPREHENSIVE AND COMBINED IN SILICO APPROACH

Objective: Inflammatory diseases have a serious impact on one’s life and represent a diverse group of ailments stemming from various causes and presenting in various forms. p38α of the mitogen-activated protein kinase family plays a crucial role in regulating inflammation, where the activation of this kinase initiates a cascade of events resulting in the production of proinflammatory mediators and cellular stress responses. In this context, attempts were made to identify potent small-molecule inhibitors of p38α and assess their binding affinity through molecular docking studies. Methods: From comprehensive reviews of several published reports, a few compounds, such as P38, P39, VPC00628, and N17, have shown substantial inhibitory activity toward p38α at various concentrations. Hence, these four compounds were chosen as lead compounds, and small-molecule libraries were constructed on the basis of their structural similarity. Next, virtual screening docking was performed to investigate the inhibitory potency of the four libraries toward the p38α isoforms (DFG-out and DFG-in), providing insights into their potential mechanisms of action. Results: In addition, a comprehensive analysis of physicochemical and pharmacokinetic properties was also performed for the identified hits from each library. Our findings have shown that, compared with those of the p38α DFG-in motif, the binding energies of the p38α DFG-out motif are greater. Conclusion: Furthermore, a few compounds from each library presented binding energies higher than those of their respective lead compounds, confirming their potential as novel therapeutic agents against inflammation.

Pimicotinib Significantly Improves Outcomes for Patients with Tenosynovial Giant Cell Tumor

Results from the Phase 3 MANEUVER trial (NCT05804045) of pimicotinib (ABSK021), an investigational orally administered, highly selective, and potent small-molecule inhibitor of colony-stimulating factor-1 receptor (CSF-1R) being developed by Abbisko Therapeutics, met its primary endpoint, demonstrating significant improvement in objective response rate (ORR) in patients with tenosynovial giant cell tumor (TGCT). The ORR for pimicotinib at week 25 was 54.0% compared with 3.2% for placebo (p<0.0001).

Pharmacophore-guided in-silico discovery of SIRT1 inhibitors for targeted cancer therapy

Epigenetic modifier, Sirtuin (SIRTs) is a family of seven isoforms (SIRT1‐7) and nicotinamide adenine dinucleotide (NAD+) dependent class III histone deacetylase (HDACs) protein. SIRT1 in association with the p53 protein can regulate crucial cell processes such as glucose metabolism, lipid metabolism, mitochondrial biogenesis, DNA repair, oxidative stress, apoptosis, and inflammation through the process of deacetylation. When SIRT1 deacetylates p53, it loses its tumor suppression property. To promote apoptosis and decrease cell proliferation by inhibiting SIRT1 protein and ultimately raising the acetylation of p53 to regain its tumor suppressor function. Though we have many SIRT1 protein inhibitors, they exhibited off-target effects and inefficiency at the clinical trial stage. This study has been executed to identify more potentially effective and reliable SIRT1 inhibitors that can perform better than the existing options. To do so, pharmacophore-based screening of compound libraries followed by virtual screening, pharmacokinetic, drug-likeness, and toxicity studies were conducted which gave 42 compounds to evaluate further. Subsequently, exhaustive molecular docking and molecular dynamics simulation predicted four potential hits to inhibit the SIRT1 protein better than the reference compound. Further studies such as principal components analysis, free energy landscape, and estimation of binding free energy were done which concluded Hit4 (PubChem ID: 55753455) to be a novel and potent SIRT1 small molecule inhibitor among the others. The total binding free energy for Hit4 was found to be −44.68 kcal/mol much better than the reference complex i.e., −29.38 kcal/mol.

Evaluation of STK17B as a cancer immunotherapy target utilizing highly potent and selective small molecule inhibitors.

The serine/threonine kinase 17B (STK17B) is involved in setting the threshold for T cell activation and its absence sensitizes T cells to suboptimal stimuli. Consequently, STK17B represents an attractive potential target for cancer immunotherapy. To assess the potential of STK17B as an immuno-oncology target, we developed potent and selective tool compounds from starting points in Blueprint Medicines Corporation's proprietary kinase inhibitor library. To characterize these molecules, enzyme and cellular assays for STK17A and STK17B were established to drive chemistry optimization. Mass spectrometry-based phosphoproteomics profiling with tool inhibitors led to the identification of Ser19 on myosin light chain 2 as STK17B substrate, which is then developed into a flow cytometry-based pharmacodynamic readout of STK17B inhibition both in vitro and in vivo. In a mouse T cell activation assay, STK17B inhibitors demonstrated the ability to enhance interleukin-2 (IL-2) production. Similarly, treatment with STK17B inhibitors resulted in stronger cytokine secretion in human T cells activated using a T cell bispecific antibody. Subsequent chemistry optimization led to the identification of a highly selective and orally bioavailable tool compound, BLU7482. In vivo, STK17B inhibition led to dose-dependent modulation of myosin light chain 2 phosphorylation and enhanced priming of naïve T cells, as determined by upregulation of CD69, IL-2 and interferon-γ secretion. In line with increased T cell activation, treatment with STK17B inhibitor enhanced antitumor activity of anti-PD-L1 antibody in the MCA205 model. In summary, we successfully identified and optimized STK17B kinase inhibitors which led to increased T cell responses in vitro and in vivo. This allowed us to evaluate the potential of STK17B inhibition as an approach for cancer immunotherapy.

7076 Inhibiting Oxidosqualene Cyclase, An Enzyme In The Cholesterol Biosynthetic Pathway, Suppresses Lung Metastasis Of Triple-Negative Breast Cancer Cells

Abstract Disclosure: Y. Liang: None. S.M. Hyder: None. Triple-negative breast cancers (TNBC) lack ER, PR and Her2neu proteins that are commonly targeted in breast cancer therapies. As a consequence, women who suffer from TNBC have poor prognosis and limited treatment options, and are usually administered toxic, non-specific chemotherapeutic agents. Drug-resistance almost always occurs, leading to tumor metastasis, the main cause of patient death. New non-toxic therapeutic strategies to control metastasis of TNBC are urgently needed. Cholesterol is an essential structural and functional component of cell membranes that is necessary for the growth of both primary tumors and metastatic colonies that reside in distant organs. Therefore, inhibiting cholesterol production is an attractive therapeutic strategy. Treatment with statins, a class of cholesterol biosynthesis inhibitors that target HMGCoA-reductase, is associated with certain undesirable side effects; consequently, we targeted oxidosqualene cyclase (OSC), an enzyme that occurs downstream of HMGCoA-reductase in the cholesterol biosynthetic pathway, to disrupt tumor metastasis. Potent small molecule inhibitors of OSC have been identified. RO 48-8071 (4′-[6-(allylmethylamino)hexyloxy]-4-bromo-2′-fluorobenzophenone fumarate) (RO), has emerged as a useful chemotherapeutic agent for treating several forms of primary tumors. We developed a mouse model for studying lung metastasis using a MDA-MB-231 variant (LM2) TNBC cell line obtained from Dr. Massagué (Memorial Sloan-Kettering Cancer Center). These are aggressive cells, and injection of only 2 X 105 cells produces detectable lung metastatic colonies in approximately 4-6 weeks. We inoculated female nu/nu mice, 5-6 weeks old, with 2 X 105 MDA-MB-231/LM2 cells in 0.1 ml DMEM/F12 medium without serum via tail vein. Five days after tumor cell inoculation animals received 20 mg/kg (n=6), 40 mg/kg (n=7) RO (ip) or vehicle alone (n=7) every other day for two weeks. Animals were then sacrificed, and both lungs harvested and immediately fixed in Bouin’s fixative. Metastatic colonies on the lung surface were identified and counted under a dissecting microscope. Our study showed that RO effectively reduced lung metastasis without affecting animal weight. Compared with 56 + 7 colonies (Mean ± SEM) in controls given vehicle alone, RO significantly reduced lung metastatic colonies to 28 ± 5 in animals exposed to 20 mg/kg and 6 ± 1 in 40 mg/kg treated animals (p<0.05, ANOVA). Reduction in number of colonies in 40 mg/kg group was also significantly different from colonies counted in 20 mg/kg treated animals. Our study is the first to demonstrate that disruption of cholesterol biosynthesis in a model of human TNBC leads to reduced metastasis in lungs. Further studies will determine mechanisms through which RO suppresses TNBC metastasis to the lungs. Supported by Zalk Missouri Professor Endowment Funds. Presentation: 6/3/2024

7076 Inhibiting Oxidosqualene Cyclase, an Enzyme in the Cholesterol Biosynthetic Pathway, Suppresses Lung Metastasis of Triple-negative Breast Cancer Cells

Abstract Disclosure: Y. Liang: None. S.M. Hyder: None. Triple-negative breast cancers (TNBC) lack ER, PR and Her2neu proteins that are commonly targeted in breast cancer therapies. As a consequence, women who suffer from TNBC have poor prognosis and limited treatment options, and are usually administered toxic, non-specific chemotherapeutic agents. Drug-resistance almost always occurs, leading to tumor metastasis, the main cause of patient death. New non-toxic therapeutic strategies to control metastasis of TNBC are urgently needed. Cholesterol is an essential structural and functional component of cell membranes that is necessary for the growth of both primary tumors and metastatic colonies that reside in distant organs. Therefore, inhibiting cholesterol production is an attractive therapeutic strategy. Treatment with statins, a class of cholesterol biosynthesis inhibitors that target HMGCoA-reductase, is associated with certain undesirable side effects; consequently, we targeted oxidosqualene cyclase (OSC), an enzyme that occurs downstream of HMGCoA-reductase in the cholesterol biosynthetic pathway, to disrupt tumor metastasis. Potent small molecule inhibitors of OSC have been identified. RO 48-8071 (4′-[6-(allylmethylamino)hexyloxy]-4-bromo-2′-fluorobenzophenone fumarate) (RO), has emerged as a useful chemotherapeutic agent for treating several forms of primary tumors. We developed a mouse model for studying lung metastasis using a MDA-MB-231 variant (LM2) TNBC cell line obtained from Dr. Massagué (Memorial Sloan-Kettering Cancer Center). These are aggressive cells, and injection of only 2 X 10[5] cells produces detectable lung metastatic colonies in approximately 4-6 weeks. We inoculated female nu/nu mice, 5-6 weeks old, with 2 X 10[5] MDA-MB-231/LM2 cells in 0.1 ml DMEM/F12 medium without serum via tail vein. Five days after tumor cell inoculation animals received 20 mg/kg (n=6), 40 mg/kg (n=7) RO (ip) or vehicle alone (n=7) every other day for two weeks. Animals were then sacrificed, and both lungs harvested and immediately fixed in Bouin’s fixative. Metastatic colonies on the lung surface were identified and counted under a dissecting microscope. Our study showed that RO effectively reduced lung metastasis without affecting animal weight. Compared with 56 + 7 colonies (Mean ± SEM) in controls given vehicle alone, RO significantly reduced lung metastatic colonies to 28 ± 5 in animals exposed to 20 mg/kg and 6 ± 1 in 40 mg/kg treated animals (p<0.05, ANOVA). Reduction in number of colonies in 40 mg/kg group was also significantly different from colonies counted in 20 mg/kg treated animals. Our study is the first to demonstrate that disruption of cholesterol biosynthesis in a model of human TNBC leads to reduced metastasis in lungs. Further studies will determine mechanisms through which RO suppresses TNBC metastasis to the lungs. Supported by Zalk Missouri Professor Endowment Funds. Presentation: 6/3/2024

CONFORMATIONAL FLEXIBILITY IS A CRITICAL FACTOR IN DESIGNING BROAD-SPECTRUM HUMAN NOROVIRUS PROTEASE INHIBITORS.

Human norovirus (HuNoV) infection is a global health and economic burden. Currently, there are no licensed HuNoV vaccines or antiviral drugs available. The protease encoded by the HuNoV genome plays a critical role in virus replication by cleaving the polyprotein and is, therefore, an excellent target for developing small molecule inhibitors. While rupintrivir, a potent small-molecule inhibitor of several picornavirus proteases, effectively inhibits GI.1 protease, it is an order of magnitude less effective against GII protease. Other GI.1 protease inhibitors also tend to be less effective against GII proteases. To understand the structural basis for the potency difference, we determined the crystal structures of proteases of GI.1, pandemic GII.4 (Houston and Sydney), and GII.3 in complex with rupintrivir. These structures show that the open substrate pocket in GI protease binds rupintrivir without requiring significant conformational changes, whereas, in GII proteases, the closed pocket flexibly extends, reorienting arginine-112 in the BII-CII loop to accommodate rupintrivir. Structures of R112A protease mutants with rupintrivir, coupled with enzymatic and inhibition studies, suggest R112 is involved in displacing both substrate and ligands from the active site, implying a role in the release of cleaved products during polyprotein processing. Thus, the primary determinant for differential inhibitor potency between the GI and GII proteases is the increased flexibility in the BII-CII loop of the GII proteases caused by H-G mutation in this loop. Therefore, the inherent flexibility of the BII-CII loop in GII proteases is a critical factor to consider when developing broad-spectrum inhibitors for HuNoV proteases.

Novel Arf1 Inhibitors Drive Cancer Stem Cell Aging and Potentiate Anti-Tumor Immunity.

The small G protein Arf1 has been identified as playing a selective role in supporting cancer stem cells (CSCs), making it an attractive target for cancer therapy. However, the current Arf1 inhibitors have limited translational potential due to their high toxicity and low specificity. In this study, two new potent small-molecule inhibitors of Arf1, identified as DU101 and DU102, for cancer therapy are introduced. Preclinical tumor models demonstrate that these inhibitors triggered a cascade of aging in CSCs and enhance anti-tumor immunity in mouse cancer and PDX models. Through single-cell sequencing, the remodeling of the tumor immune microenvironment induced by these new Arf1 inhibitors is analyzed and an increase in tumor-associated CD8+ CD4+ double-positive T (DPT) cells is identified. These DPT cells exhibit superior features of active CD8 single-positive T cells and a higher percentage of TCF1+PD-1+, characteristic of stem-like T cells. The frequency of tumor-infiltrating stem-like DPT cells correlates with better disease-free survival (DFS) in cancer patients, indicating that these inhibitors may offer a novel cancer immunotherapy strategy by converting the cold tumor immune microenvironment into a hot one, thus expanding the potential for immunotherapy in cancer patients.

Phosphoenolpyruvate carboxykinase-2 (PCK2) is a therapeutic target in triple-negative breast cancer.

Metabolic rewiring in malignant transformation is often accompanied by altered expression of metabolic isozymes. Phosphoenolpyruvate carboxykinase-2 (PCK2) catalyzes the rate-limiting step of gluconeogenesis and is the dominant isoform in many cancers including triple-negative breast cancer (TNBC). Our goal was to identify small molecule inhibitors of PCK2 enzyme activity. We assessed the impact of PCK2 down regulation with shRNA on TNBC cell growth in vitro and used AtomNet® deep convolutional neural network software to identify potential small molecule inhibitors of PCK2-based structure. We iteratively tested candidate compounds in an in vitro PCK-2 enzyme assay. The impact of the top hit on metabolic flux and cell viability was also assessed. PCK2 downregulation decreased growth of BT-549 and MDA-MB-231 cells and reduced metabolic flux through pyruvate carboxylase. The first AtomNet® in silico structural screen of 7 million compounds yielded 86 structures that were tested in PCK2 enzyme assay in vitro. The top hit (IC50 = 2.4µM) was used to refine a second round of in silico screen that yielded 82 candidates to be tested in vitro, which resulted in 45 molecules with inhibition > 20%. In the second in vitro screen we also included 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate, previously suggested to be PCK2 inhibitor based on structure, which emerged as the top hit. The specificity of this compound was tested in PCK1 and PCK2 enzymatic assays and showed IC50 of 500nM and 3.5-27nM for PCK1 and PCK2, respectively. 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate is a high affinity PCK2 enzyme inhibitor that also has significant growth inhibitory activity in breast cell lines in vitro and represents a potential therapeutic lead compound.

Dengue virus: pathogenesis and potential for small molecule inhibitors.

Dengue, caused by dengue virus (DENV), is now endemic in nearly 100 countries and infection incidence is reported in another 30 countries. Yearly an estimated 400 million cases and 2200 deaths are reported. Effective vaccines against DENV are limited and there has been significant focus on the development of effective antiviral against the disease. The World Health Organization has initiated research programs to prioritize the development and optimization of antiviral agents against several viruses including Flaviviridae. A significant effort has been taken by the researchers to develop effective antivirals against DENV. Several potential small-molecule inhibitors like efavirenz, tipranavir and dasabuvir have been tested against envelope and non-structural proteins of DENV, and are in clinical trials around the world. We recently developed one small molecule, namely 7D, targeting the host PF4-CXCR3 axis. 7D inhibited all 4 serotypes of DENV in vitro and specifically DENV2 infection in two different mice models. Although the development of dengue vaccines remains a high priority, antibody cross reactivity among the serotypes and resulting antibody-dependent enhancement (ADE) of infection are major concerns that have limited the development of effective vaccine against DENV. Therefore, there has been a significant emphasis on the development of antiviral drugs against dengue. This review article describes the rescue effects of some of the small molecule inhibitors to viral/host factors associated with DENV pathogenesis.

Construction of IRAK4 inhibitor activity prediction model based on machine learning.

Interleukin-1 receptor-associated kinase 4 (IRAK4) is a crucial serine/threonine protein kinase that belongs to the IRAK family and plays a pivotal role in Toll-like receptor (TLR) and Interleukin-1 receptor (IL-1R) signaling pathways. Due to IRAK4's significant role in immunity, inflammation, and malignancies, it has become an intriguing target for discovering and developing potent small-molecule inhibitors. Consequently, there is a pressing need for rapid and accurate prediction of IRAK4 inhibitor activity. Leveraging a comprehensive dataset encompassing activity data for 1628 IRAK4 inhibitors, we constructed a prediction model using the LightGBM algorithm and molecular fingerprints. This model achieved an R2 of 0.829, an MAE of 0.317, and an RMSE of 0.460 in independent testing. To further validate the model's generalization ability, we tested it on 90 IRAK4 inhibitors collected in 2023. Subsequently, we applied the model to predict the activity of 13,268 compounds with docking scores less than -9.503kcal/mol. These compounds were initially screened from a pool of 1.6 million molecules in the chemdiv database through high-throughput molecular docking. Among these, 259 compounds with predicted pIC50 values greater than or equal to 8.00 were identified. We then performed ADMET predictions on these selected compounds. Finally, through a rigorous screening process, we identified 34 compounds that adhere to the four complementary drug-likeness rules, making them promising candidates for further investigation. Additionally, molecular dynamics simulations confirmed the stable binding of the screened compounds to the IRAK4 protein. Overall, this work presents a machine learning model for accurate prediction of IRAK4 inhibitor activity and offers new insights for subsequent structure-guided design of novel IRAK4 inhibitors.