Potent Small Molecule Inhibitor Research Articles

Specific Inhibitors of Mitochondrial Deacylase Sirtuin 4 Endowed with Cellular Activity.

Sirtuins are NAD+-dependent protein lysine deacylases implicated in aging-related diseases. Mammalian Sirtuin 4 (Sirt4) is located in mitochondria and a potential therapeutic target for cancer and metabolic diseases, but no potent and selective Sirt4 inhibitors have been reported. Here, we describe the identification of potent Sirt4-specific small-molecule inhibitors. Testing hits from a target-based virtual screen revealed 12 active compounds. A focused screen based on two top compounds, followed by structure-assisted design of derivatives, yielded four first-in-class potent Sirt4 inhibitors. Kinetic analyses indicate compound competition with the acyl peptide substrate, consistent with the docking models and implicating Sirt4's unique acyl binding site. The compounds indeed show preference for Sirt4 over other isoforms, with one of them (69) being highly isoform selective, and they are active in cells. Our results provide first lead compounds and mechanistic insights for optimization toward Sirt4-specific inhibitors useful as experimental tools and potential therapeutics.

Virtual screening of flavonoids as potential RIPK1 inhibitors for neurodegeneration therapy.

Global prevalence of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease is increasing gradually, whereas approvals of successful therapeutics for central nervous system disorders are inadequate. Accumulating evidence suggests pivotal roles of the receptor-interacting serine/threonine-protein kinase 1 (RIPK1) in modulating neuroinflammation and necroptosis. Discoveries of potent small molecule inhibitors for RIPK1 with favorable pharmacokinetic properties could thus address the unmet medical needs in treating neurodegeneration. In a structure-based virtual screening, we performed site-specific molecular docking of 4,858 flavonoids against the kinase domain of RIPK1 using AutoDock Vina. We predicted physicochemical descriptors of the top ligands using the SwissADME webserver. Binding interactions of the best ligands and the reference ligand L8D were validated using replicated 500-ns Gromacs molecular dynamics simulations and free energy calculations. From Vina docking, we shortlisted the top 20 flavonoids with the highest binding affinities, ranging from -11.7 to -10.6 kcal/mol. Pharmacokinetic profiling narrowed down the list to three orally bioavailable and blood-brain-barrier penetrant flavonoids: Nitiducarpin, Pinocembrin 7-O-benzoate, and Paratocarpin J. Next, trajectories of molecular dynamics simulations of the top protein-ligand complexes were analyzed for binding interactions. The root-mean-square deviation (RMSD) was 1.191 Å (±0.498 Å), 1.725 Å (±0.828 Å), 1.923 Å (±0.942 Å), 0.972 Å (±0.155 Å) for Nitiducarpin, Pinocembrin 7-O-benzoate, Paratocarpin J, and L8D, respectively. The radius of gyration (Rg) was 2.034 nm (±0.015 nm), 2.0.39 nm (± 0.025 nm), 2.053 nm (±0.021 nm), 2.037 nm (±0.016 nm) for Nitiducarpin, Pinocembrin 7-O-benzoate, Paratocarpin J, and L8D, respectively. The solvent accessible surface area (SASA) was 159.477 nm2 (±3.021 nm2), 159.661 nm2 (± 3.707 nm2), 160.755 nm2 (±4.252 nm2), 156.630 nm2 (±3.521 nm2), for Nitiducarpin, Pinocembrin 7-O-benzoate, Paratocarpin J, and L8D complexes, respectively. Therefore, lower RMSD, Rg, and SASA values demonstrated that Nitiducarpin formed the most stable complex with the target protein among the best three ligands. Finally, 2D protein-ligand interaction analysis revealed persistent hydrophobic interactions of Nitiducarpin with the critical residues of RIPK1, including the catalytic triads and the activation loop residues, implicated in the kinase activity and ligand binding. Our target-based virtual screening identified three flavonoids as strong RIPK1 inhibitors, with Nitiducarpin exhibiting the most potent inhibitory potential. Future in vitro and in vivo studies with these ligands could offer new hope for developing effective therapeutics and improving the quality of life for individuals affected by neurodegeneration.

ARC-8: Phase 1/1b randomized study of quemliclustat + gemcitabine/nab-paclitaxel ± zimberelimab in patients with treatment-naive metastatic pancreatic adenocarcinoma.

665 Background: Metastatic pancreatic ductal adenocarcinoma (mPDAC) remains a challenging cancer to treat, with median survival of <1 year. In PDAC, extracellular adenosine (eADO) produced from ATP is released from tumor cells and suppresses antitumor immune responses. Quemliclustat (Q) is a potent and selective small-molecule inhibitor of soluble and cell-bound CD73, a key enzyme involved in the production of eADO within the tumor microenvironment. Elevated CD73 expression is found in 40-60% of PDAC and correlates with poor clinical outcome. ARC-8 evaluated Q ± anti-PD-1 antibody zimberelimab (Z) in combination with gemcitabine/nab-paclitaxel (G/nP). Methods: ARC-8 (NCT04104672) is a phase 1b dose escalation and expansion study in patients (pts) with treatment-naive mPDAC. Q 100mg was previously determined as the recommended dose for expansion (Manji et al., 2021). Q 100 mg is administered with standard doses of G/nP with Z (240 mg IV Q2W) in the dose expansion (Cohort A; QZ+G/nP) and in randomized (2:1) cohorts either with Z (Cohort A1; QZ+G/nP) or without Z (Cohort A2; Q+G/nP). Adverse events (AEs) are recorded and graded per NCI CTCAE 5.0. Clinical activity is assessed every 8 weeks per RECIST v1.1. Endpoints include measures of safety and median overall survival (OS). Results: As of June 19, 2023, 122 patients with untreated mPDAC were treated with Q 100mg, including 93 pts treated with QZ+G/nP (dose escalation, n=6; cohort A, n=26; and randomized cohort A1, n=61) and 29 pts treated with Q+G/nP (Cohort A2). Median age was 66 years, 66% ECOG of 1, and 65% had liver metastasis at baseline. Median survival follow-up was 21 months (mos). Efficacy is summarized in Table 1. All patients reported AE, 85% Grade 3+ AE, and 23% discontinued study treatment due to AE. Most common Grade 3+ AE was neutrophil count decreased (31%) and anemia (25%). Conclusions: Results from ARC-8 demonstrate the addition of Q 100 mg ± Z to G/nP was safe and tolerable, with no significant added toxicity to GnP. Modulation of eADO with Q may confer benefit beyond radiographic measures of disease. The OS is promising and supports further development of Q in mPDAC. Clinical trial information: NCT04104672 . [Table: see text]

Molecular dynamics simulation study on the binding mechanism between carbon nanotubes and RNA-dependent RNA polymerase

Carbon nanotubes (CNTs) have potential prospects in disease treatment, so it is of great significance to study CNTs as the possible inhibitors of RNA-dependent RNA polymerase (RdRp). Through the way of using the RdRp of SARS-COV-2 as a model, five armchair single-walled carbon nanotubes (SWCNTs) (namely Dn, which stands for CNTs (n, m = n), n = 3–7) and RdRp have been selected to study the interactions by means of molecular docking and molecular dynamics simulation. After five SWCNT-RdRp complex systems have been subjected to the molecular dynamics simulations of 100 ns, and Molecular Mechanics Poisson − Boltzmann Surface Area (MMPBSA) has been used to calculate the binding free energy, it is found that the binding free energy of the D6 system (-189.541 kJ/mol) is significantly higher than that of the other four systems, and most of the amino acids with strong positive effects on binding are usually basic amino acids. What’s more, in the further investigation of the specific interaction mechanism between CNT (6,6) and RdRp, it is revealed that the three amino acid residues LYS545, ARG553 and ARG555 located in the nucleoside triphosphate (NTP) entry channel all have strong effects. In addition, it is also observed that when ARG555 has been inserted into SWCNT, a stable structure will be formed, which will break the original NTP entry channel structure and inhibit virus replication. Therefore, it can be concluded that certain specific types of SWCNT, such as CNT (6,6), could be potential small molecule inhibitors in the treatment of coronavirus. Communicated by Ramaswamy H. Sarma

Abstract A078: Response and resistance to KRAS inhibition in PDAC mouse models

Abstract Pancreatic ductal adenocarcinoma (PDAC), among the most lethal cancers with a 5-year survival rate of 11%, is a KRAS-driven disease. Studies have shown that a single glycine (G) to aspartic acid (D) substitution on codon 12 of KRAS protein (known as KRASG12D mutation) appears in 41% of all PDAC cases, highlighting the necessity of targeting KRASG12D as a therapeutic approach. MRTX1133, a recently developed potent, non-covalent, direct, and selective KRASG12D small molecule inhibitor demonstrates a high-affinity interaction with KRASG12D. Previous studies have shown striking efficacy of MRTX1133 on growth inhibition of mouse/human PDAC tumor cells carrying KRASG12D mutation in various in vivo and in vitro models, during short-term treatment. Here in our study, we investigated the efficacy of continuous long-term treatment with MRTX1133 in two different PDAC mouse models: KPC (a genetically engineered mouse model harboring a KRASG12D mutation) and its syngeneic orthotopic model. In both models, tumor sizes were detected by ultrasound imaging and after reaching 100-300 mm3 in volume, mice were enrolled in either MRTX1133 or vehicle treatment (30mg/kg, IP, BID). Tumor sizes were monitored by ultrasound regularly and the treatment continued until mice reached clinical endpoint. At endpoint, mice were sacrificed, and tumors were harvested for a series of downstream experiments. Our results showed that while, in both models, vehicle treated tumors grew aggressively and mice reached endpoint in average 6 days post treatment, drug treated tumors regressed rapidly and survival rate increased significantly to as late as 222 days post treatment in KPC model. However, in both models, drug treated tumors ultimately relapsed, developed resistance to the drug, and resulted in death. Further analysis of KPC mice ultrasound images evidenced that the relapsed tumors occurred at the same sites of the primary tumors. This suggests the possibility of relapsed tumors arising from the regrowth of the dormant residual cells from the primary tumors. Re-implantation of the resistant tumors either in the flank or pancreata of the new wildtype mice resulted in the formation of drug-resistant tumors in hosts. In addition, histopathology analysis of the tumor sections demonstrated a significant increase in the ratio of invasive PDAC to PanIN areas in the drug resistant tumors. Furthermore, multiplex immunofluorescence of both MRTX1133 resistant and vehicle treated tumors from KPC mice demonstrated co-expression of markers representing basal-like and classical PDAC subtypes. Moreover, flow cytometry analysis indicated an increase in the myeloid population of the resistant tumors comparing to the vehicle treated group. In the future directions, through applying a broad range of cellular and molecular experiments we will characterize the kinetics of KRAS inhibitor drug resistance and define possible combination therapy startegies to overcome drug resistance mechanisms. Citation Format: Laleh Abbassi, Jullien Dilly, Annan Yang, Giselle Uribe, Branden Parent, Connor Hennessey, Kevin Kapner, Alexander Jordan, Shatavisha Dasgupta, Micaela Morgado, Taimour Baslan, Li Qiang, Peter Winter, Seema Chugh, Alex Shalek, Ben Stanger, Srivastan Raghavan, Scott Lowe, Brian Wolpin, Stephanie Dougan, Jonathan Nowak, Andrew Aguirre. Response and resistance to KRAS inhibition in PDAC mouse models [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A078.

Mutational analysis of SARS-CoV-2 ORF6-KPNA2 binding interface and identification of potent small molecule inhibitors to recuse the host immune system.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) surfaced on 31 December, 2019, and was identified as the causative agent of the global COVID-19 pandemic, leading to a pneumonia-like disease. One of its accessory proteins, ORF6, has been found to play a critical role in immune evasion by interacting with KPNA2 to antagonize IFN signaling and production pathways, resulting in the inhibition of IRF3 and STAT1 nuclear translocation. Since various mutations have been observed in ORF6, therefore, a comparative binding, biophysical, and structural analysis was used to reveal how these mutations affect the virus's ability to evade the human immune system. Among the identified mutations, the V9F, V24A, W27L, and I33T, were found to have a highly destabilizing effect on the protein structure of ORF6. Additionally, the molecular docking analysis of wildtype and mutant ORF6 and KPNA2 revealed the docking score of - 53.72 kcal/mol for wildtype while, -267.90 kcal/mol, -258.41kcal/mol, -254.51 kcal/mol and -268.79 kcal/mol for V9F, V24A, W27L, and I33T respectively. As compared to the wildtype the V9F showed a stronger binding affinity with KPNA2 which is further verified by the binding free energy (-42.28 kcal/mol) calculation. Furthermore, to halt the binding interface of the ORF6-KPNA2 complex, we used a computational molecular search of potential natural products. A multi-step virtual screening of the African natural database identified the top 5 compounds with best docking scores of -6.40 kcal/mol, -6.10 kcal/mol, -6.09 kcal/mol, -6.06 kcal/mol, and -6.03 kcal/mol for tophit1-5 respectively. Subsequent all-atoms simulations of these top hits revealed consistent dynamics, indicating their stability and their potential to interact effectively with the interface residues. In conclusion, our study represents the first attempt to establish a foundation for understanding the heightened infectivity of new SARS-CoV-2 variants and provides a strong impetus for the development of novel drugs against them.

Structure-Guided Design, Synthesis, and Antivirulence Assessment of Covalent Staphylococcus aureus Sortase A Inhibitors.

Sortase A (SrtA) is a membrane-associated cysteine transpeptidase required for bacterial virulence regulation and anchors surface proteins to cell wall, thereby assisting biofilm formation. SrtA is targeted in antivirulence treatments against Gram-positive bacterial infections. However, the development of potent small-molecule SrtA inhibitors is constrained owing to the limited understanding of the mode of action of inhibitors in the SrtA binding pocket. Herein, we designed and synthesized a novel class of covalent SrtA inhibitors based on the binding mode detailed in the X-ray crystal structure of the ML346/Streptococcus pyogenes SrtA complex. ML346 analog Y40 exhibited 2-fold increased inhibitory activity on Staphylococcus aureus SrtA and showed superior inhibitory effects on biofilm formation in vitro. Y40 protected Galleria mellonella larvae fromS. aureusinfections in vivo while minimally attenuating staphylococcal growth in vitro. Our study indicates that the covalent SrtA inhibitor Y40 is an antivirulence agent that is effective againstS. aureusinfections.

EZH2 inhibitors promote β-like cell regeneration in young and adult type 1 diabetes donors

β-cells are a type of endocrine cell found in pancreatic islets that synthesize, store and release insulin. In type 1 diabetes (T1D), T-cells of the immune system selectively destroy the insulin-producing β-cells. Destruction of these cells leads to a lifelong dependence on exogenous insulin administration for survival. Consequently, there is an urgent need to identify novel therapies that stimulate β-cell growth and induce β-cell function. We and others have shown that pancreatic ductal progenitor cells are a promising source for regenerating β-cells for T1D owing to their inherent differentiation capacity. Default transcriptional suppression is refractory to exocrine reaction and tightly controls the regenerative potential by the EZH2 methyltransferase. In the present study, we show that transient stimulation of exocrine cells, derived from juvenile and adult T1D donors to the FDA-approved EZH2 inhibitors GSK126 and Tazemetostat (Taz) influence a phenotypic shift towards a β-like cell identity. The transition from repressed to permissive chromatin states are dependent on bivalent H3K27me3 and H3K4me3 chromatin modification. Targeting EZH2 is fundamental to β-cell regenerative potential. Reprogrammed pancreatic ductal cells exhibit insulin production and secretion in response to a physiological glucose challenge ex vivo. These pre-clinical studies underscore the potential of small molecule inhibitors as novel modulators of ductal progenitor differentiation and a promising new approach for the restoration of β-like cell function.

The small-molecule drug homoharringtonine targets HSF1 to suppress pancreatic cancer progression.

Heat shock factor 1 (HSF1), an essential transcription factor for stress response, is exploited by various tumors to facilitate their initiation, progression, invasion, and migration. Amplification of HSF1 is widely regarded as an indicator in predicting cancer severity, the likelihood of treatment failure and reduced patient survival. Notably, HSF1 is markedly amplified in 40% of pancreatic cancer (PC), which typically have limited treatment options. HSF1 has been proven to be a promising therapeutic target for multiple cancers. However, a direct small molecule HSF1 inhibitor with sufficient bioactivity and reliable safety has not been developed clinically. In this study, we successfully established a high-throughput screening system utilizing luciferase reporter assay specifically designed for HSF1, which leads to the discovery of a potent small molecule inhibitor targeting HSF1. Homoharringtonine (HHT) selectively inhibited PC cell viability with high HSF1 expression and induced a markedly stronger tumor regression effect in the subcutaneous xenograft model than the comparator drug KRIBB11, known for its direct action on HSF1. Moreover, HHT shows promise in countering the resistance encountered with HSP90 inhibitors, which have been observed to increase heat shock response intensity in clinical trials. Mechanistically, HHT directly bound to HSF1, suppressing its expression and thereby inhibiting transcription of HSF1 target genes. In conclusion, our work presents a preclinical discovery and validation for HHT as a HSF1 inhibitor for PC treatment.

A novel TREX1 inhibitor, VB-85680, upregulates cellular interferon responses.

Activation of the cGAS-STING pathway plays a key role in the innate immune response to cancer through Type-1 Interferon (IFN) production and T cell priming. Accumulation of cytosolic double-stranded DNA (dsDNA) within tumor cells and dying cells is recognized by the DNA sensor cyclic GMP-AMP synthase (cGAS) to create the secondary messenger cGAMP, which in turn activates STING (STimulator of INterferon Genes), resulting in the subsequent expression of IFN-related genes. This process is regulated by Three-prime Repair EXonuclease 1 (TREX1), a 3' → 5' exonuclease that degrades cytosolic dsDNA, thereby dampening activation of the cGAS-STING pathway, which in turn diminishes immunostimulatory IFN secretion. Here, we characterize the activity of VB-85680, a potent small-molecule inhibitor of TREX1. We first demonstrate that VB-85680 inhibits TREX1 exonuclease activity in vitro in lysates from both human and mouse cell lines. We then show that treatment of intact cells with VB-85680 results in activation of downstream STING signaling, and activation of IFN-stimulated genes (ISGs). THP1-Dual™ cells cultured under low-serum conditions exhibited an enhanced ISG response when treated with VB-85680 in combination with exogenous DNA. Collectively, these findings suggest the potential of a TREX1 exonuclease inhibitor to work in combination with agents that generate cytosolic DNA to enhance the acquisition of the anti-tumor immunity widely associated with STING pathway activation.

Development of MPS1 Inhibitors: Recent Advances and Perspectives.

Monopolar spindle kinase 1 (MPS1) plays a pivotal role as a dual-specificity kinase governing spindle assembly checkpoint activation and sister chromatid separation in mitosis. Its overexpression has been observed in various human malignancies. MPS1 reduces spindle assembly checkpoint sensitivity, allowing tumor cells with a high degree of aneuploidy to complete mitosis and survive. Thus, MPS1 has emerged as a promising candidate for cancer therapy. Despite the identification of numerous MPS1 inhibitors, only five have advanced to clinical trials with none securing FDA approval for cancer treatment. In this perspective, we provide a concise overview of the structural and functional characteristics of MPS1 by highlighting its relevance to cancer. Additionally, we explore the structure-activity relationships, selectivity, and pharmacokinetics of MPS1 inhibitors featuring diverse scaffolds. Moreover, we review the reported work on enhancing MPS1 inhibitor selectivity, offering valuable insights into the discovery of novel, highly potent small-molecule MPS1 inhibitors.

High-throughput virtual screening to identify potential small molecule inhibitors of the Zα domain of the adenosine deaminases acting on RNA 1(ADAR1)

Changes in RNA editing are closely associated with diseases such as cancer, viral infections, and autoimmune disorders. Adenosine deaminase (ADAR1), which acts on RNA 1, plays a key role in adenosine to inosine editing and is a potential therapeutic target for these various diseases. The p150 subtype of ADAR1 is the only one that contains a Zα domain that binds to both Z-DNA and Z-RNA. The Zα domain modulates immune responses and may be suitable targets for antiviral therapy and cancer immunotherapy. In this study, we attempted to utilize molecular docking to identify potential inhibitors that bind to the ADAR1 Zα domain. The virtual docking method screened the potential activity of more than 100,000 compounds on the Zα domain of ADAR1 and filtered to obtain the highest scoring results.We identified 71 compounds promising to bind to ADAR1 and confirmed that two of them, lithospermic acid and Regaloside B, interacts with the ADAR1 Zα domain by surface plasmonic resonance technique. The molecular dynamics calculation of the complex of lithospermic acid and ADAR1 also showed that the binding effect of lithospermic acid to ADAR1 was stable.This study provides a new perspective for the search of ADAR1 inhibitors, and further studies on the anti-ADAR11 activity of these compounds have broad prospects.

Establishment and characterization of a patient-derived solitary fibrous tumor/hemangiopericytoma cell line model.

Solitary fibrous tumor/Hemangiopericytoma (SFT/HPC) is a rare subtype of soft tissue sarcoma harboring NAB2-STAT6 gene fusions. Mechanistic studies and therapeutic development on SFT/HPC are impeded by scarcity and lack of system models. In this study, we established and characterized a novel SFT/HPC patient-derived cell line (PDC), SFT-S1, and screened for potential drug candidates that could be repurposed for the treatment of SFT/HPC. Immunohistochemistry profiles of the PDC was consistent with the patient's tumor sample (CD99+/CD34+/desmin-). RNA sequencing, followed by Sanger sequencing confirmed the pathognomonic NAB2exon3-STAT6exon18 fusion in both the PDC and the original tumor. Transcriptomic data showed strong enrichment for oncogenic pathways (epithelial-mesenchymal transition, FGF, EGR1 and TGFβ signaling pathways) in the tumor. Whole genome sequencing identified potentially pathogenic somatic variants such as MAGEA10 and ABCA2. Among a panel of 14 targeted agents screened, dasatinib was identified to be the most potent small molecule inhibitor against the PDC (IC50, 473 nM), followed by osimertinib (IC50, 730 nM) and sunitinib (IC50, 1765 nM). Methylation profiling of the tumor suggests that this specific variant of SFT/HPC could lead to genome-wide hypomethylation. In conclusion, we established a novel PDC model of SFT/HPC with comprehensive characterization of its genomic, epigenomic and transcriptomic landscape, which can facilitate future preclinical studies of SFT/HPC, such as in vitro drug screening and in vivo drug testing.

Abstract C160: HMPL-653, a highly potent and selective CSF-1R inhibitor, targeting both tumor cells and tumor microenvironment

Abstract Background: Colony stimulating factor 1 receptor (CSF-1R) and its ligand CSF-1 (CSF-1R/CSF-1) signaling regulates the function and survival of tumor-associated macrophages (TAM), which are involved in tumor progression and suppression of anti-tumor immunity. Moreover, activation of CSF-1R/CSF-1 axis including CSF-1 fusions or CSF-1R activating mutations has also been implicated in the pathogenesis of certain tumors such as tenosynovial giant cell tumor and histiocytic neoplasms. HMPL-653 is a highly potent and selective CSF-1R small molecule inhibitor, discovered and being currently developed in phase I clinical trial (NCT05277454) by HUTCHMED. Methods: The inhibition on CSF-1R kinase was determined using Z-LYTE™ kinase assay. The selectivity of HMPL-653 was assessed against a panel of 413 kinases by Eurofins. Cellular target inhibition on phosphorylation of CSF-1R (p-CSF-1R) was detected by ELISA. In vitro cell viability was measured by CCK-8 assay. Multiple CSF-1/CSF-1R driven tumor models in Nu/Nu nude mice were applied to determine the anti-tumor activity of HMPL-653 as a single agent. To demonstrate the immunoregulatory effect of HMPL-653, a murine tumor model, B16F10, was established in C57BL/6J mice, and the combination regimen, HMPL-653 plus PCP (poly (I:C)+CpG+anti-PD-1) was evaluated. Results: HMPL-653 strongly inhibited CSF-1R kinase with IC50 of 5 nM, showing >7-fold potency than that of the approved CSF-1R inhibitor pexidartinib (IC50=39 nM). And the selectivity profiling in 413 kinase panels revealed that HMPL-653 was highly potent only against CSF-1R, indicating a lower off-target risk than paxidatinib reported. HMPL-653 potently inhibited CSF-1 stimulated p-CSF-1R in THP-1 cells with IC50 of 5 nM. In CSF-1/CSF-1R-dependent cell lines, M-NFS-60 (mouse myelogenous leukemia) and three cell lines stably expressed BCR-CSF-1R or CSF-1R mutations (Ba/F3-BCR-CSF-1R, Ba/F3-CSF-1RW450-E456del, and Ba/F3-CSF-1RY546-K551del), HMPL-653 significantly down regulated p-CSF-1R and its downstream signaling p-ERK and p-AKT. As a consequence, HMPL-653 robustly inhibited the cell viability of above cell models (IC50s: 3~40 nM). Besides the direct anti-tumor effect, HMPL-653 also demonstrated strong inhibition on macrophage survival and M2 macrophage polarization, indicating a potential effect on targeting tumor microenvironment. In vivo, oral administration of HMPL-653 induced remarkable and dose-dependent anti-tumor efficacies in M-NFS-60 ascites model and subcutaneous tumor models harboring above mentioned CSF-1R alterations. And the PK/PD analysis revealed that the anti-tumor effect correlated well with p-CSF-1R inhibition. Moreover, HMPL-653 significantly improved the anti-tumor activity of PCP regimen in B16F10 model by reducing TAMs including M2 macrophages and increasing the ratio of M1/M2 macrophages. Conclusion: HMPL-653 is a highly potent and selective CSF-1R small molecule inhibitor which targets both CSF-1/CSF-1R-dependent tumors and tumor associated macrophages, warranting further clinical evaluation. Citation Format: Jia Hu, Liang Ge, Hui Zhang, An Jiang, Juntao Yu, Weifang Xue, Jian Wang, Yang Sai, Na Yang, Weiguo Qing, Yongxin Ren, Michael Shi, Weiguo Su. HMPL-653, a highly potent and selective CSF-1R inhibitor, targeting both tumor cells and tumor microenvironment [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 C160.

Abstract C095: The menin inhibitor VTP-50469 enhances the in vivo efficacy of established drugs against preclinical models of aggressive infant MLL-r acute lymphoblastic leukemia

Abstract Introduction: Over 70% of infants with acute lymphoblastic leukemia (ALL) present with mixed lineage leukemia (MLL1/KMT2A) gene rearrangements (MLL-r ALL), which is associated with poor prognosis. MLL-r encodes oncogenic fusion proteins that cause aberrant chromatin histone methylation and leukemic transformation of hematopoietic progenitors. A characteristic of MLL-r leukemia is high expression of the FLT3 gene. Menin is critical for leukemic transformation by MLL fusion proteins. VTP-50469, the preclinical precursor of revumenib (SNDX-5613), is a potent and selective small molecule inhibitor of the Menin-MLL interaction that has shown impressive single-agent activity in preclinical models of MLL-r acute leukemia. Revumenib is currently undergoing clinical evaluation, therefore it was of interest to assess the in vivo activity of VTP-50469 in combination with established drugs against MLL-r ALL patient-derived xenografts (PDXs). Methods: VTP-50469 was tested as a single agent against a panel of 7 pediatric MLL-r ALL PDXs in immune-deficient (NSG) mice (120 mg/kg twice daily x 28 days via oral gavage). Combination studies used VTP-50469 formulated in chow (0.1%) available ad libitum for 28 days in combination with either an induction-type regimen of vincristine (0.15 mg/kg once weekly x 4 weeks), dexamethasone (5 mg/kg) and L-asparaginase (1,000 U/kg) both 5 days on 2 days off x 4 weeks, VXL) all administered intraperitoneally, or the FLT3 inhibitor gilteritinib (Xospata, 30 mg/kg x 28 days via oral gavage). Events were defined as >25% huCD45+ in the peripheral blood. Drug efficacy was assessed by event-free survival of treated (T) and control (C) groups by T-C, T/C measurements and stringent objective response criteria (progressive disease, PD; complete response, CR; maintained CR, MCR), and disease infiltration in the spleen and femoral bone marrow (BM) at 28 days post-treatment initiation. Results: Single-agent VTP-50469 elicited MCRs in 6/7 PDXs and significantly (P<0.0001) reduced leukemia infiltration of the spleen (7/7 PDXs) and BM (6/7 PDXs) compared to vehicle control mice. When administered in chow, VTP-50469 elicited a CR in the MLL-7 PDX (T-C 48.2 days, T/C 8.1) and a PD in MLL-8 (T-C 23.0 days, T/C 4.9). VTP-50469 combined with VXL elicited MCRs in both PDXs (MLL-7 T-C 77.0 days, T/C 12.3; MLL-8 T-C 49.1 days, T/C 9.3) and significantly delayed leukemia progression compared with VTP-50469 or VXL alone (P=0.0013). Moreover, VTP-50469 combined with VXL almost completely eradicated leukemia infiltration of spleens and BMs by both PDXs. VTP-50469 combined with gilteritinib did not result in therapeutic enhancement compared to each single agent arm in either PDX. Conclusions: The addition of VTP-50469 to a standard-of-care induction-type regimen (VXL) resulted in therapeutic enhancement against two infant MLL-r ALL PDXs, while VTP-50469 plus gilteritinib was substantially less effective. These results support further clinical evaluation of revumenib in combination with standard-of-care therapy in MLL-r leukemia. Citation Format: Richard B Lock, Kathryn Evans, Sophia Gawan-Taylor, Ben Watts, Timothy Stearns, Eric J Earley, Emily L Jocoy, Carol J Bult, Beverly A Teicher, Gerard M McGeehan, Malcolm A Smith. The menin inhibitor VTP-50469 enhances the in vivo efficacy of established drugs against preclinical models of aggressive infant MLL-r acute lymphoblastic leukemia [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 C095.

Abstract A024: Preclinical profile of novel and potent small molecule inhibitors of KIF18A inhibitors in chromosomally unstable solid tumor lines

Abstract Accurate alignment of chromosomes at the equator region and equal chromosome separation during cell division is essential for maintenance of genome integrity. KIF18A belongs to kinesin superfamily of microtubule associated motor proteins and is responsible for maintenance of bipolar spindle fiber integrity during mitotic phase. Cancer cells with chromosomally unstable (CIN+) DNA show synthetic lethal phenotype with genetic ablation of KIF18A. Thus, pharmacological inhibition of KIF18A represents a potential strategy aimed at attenuating growth of CIN +ve tumors. A microtubule dependent ADP Glo kinase assay was used to identify potent KIF18A inhibitors. Mitotic arrest, manifested by an increase in phosphoH3, was determined in cells using fluorescent microscopy. Anti-proliferative activity was confirmed in CIN+ve solid tumor cells with significant selectivity over CIN-ve cancer cells. Compounds inhibited KIF18A at with potencies ranging from 50-80 nM. A dose dependent increase in mitotic index in CIN+ve OVCAR-3 cells (via fluorescence microscopy) was observed in contrast to CIN-ve HCT-116 cells (10-100X selectivity). Translation was evident in an antiproliferation assay using CIN-ve cancer cells with IC50 <10 nM. Selected KIF18A inhibitors demonstrated favorable physicochemical, ADME and PK properties. In Vivo efficacy studies are planned. Citation Format: Sukanya Patra, Manoj Pothuganti, Navnath Karche, Ganesh Gudade, Venu Sankeshi, Githavani Kummari, Srikant Vishwanadha. Preclinical profile of novel and potent small molecule inhibitors of KIF18A inhibitors in chromosomally unstable solid tumor lines [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 A024.

High-throughput virtual screening of potential inhibitors of GPR52 using docking and biased sampling method for Huntington's disease therapy.

Huntington's disease (HD) is a rare and progressive neurodegenerative disorder caused by polyglutamine (poly-Q) mutations of the huntingtin (HTT) gene resulting in chorea, cognitive, and psychiatric dysfunctions. Being a monogenic condition, reducing the levels of the mutated huntingtin protein (mHTT) holds promise as an effective therapeutic approach. GPR52, an orphan G-protein coupled receptor (GPCR), enriched in the striatum, is a novel target for slowing down the progression of HD by lowering the mHTT levels. Therefore, the study focuses on identifying potent small-molecule inhibitors for GPR52 using a combination of robust high-throughput virtual screening (HTVS) and pharmacokinetics profiling followed by fast pulling of ligand (FPL) and umbrella sampling (US) simulations. Initially, screening a library of 2,36,545 compounds was done against the binding pocket of GPR52. Based on binding affinity, stereochemical and non-bonded interactions, and pharmacokinetic profiling, 50 compounds were shortlisted. Selected hit compounds 1, 2, and 3 were subjected to FPL simulations with applied external bias potential to investigate their unique dissociation pathways and intermolecular interactions over time. Subsequently, the US simulations were performed on the selected hit compounds to estimate their binding free energy (ΔG). The analysis of the trajectories obtained from simulations revealed that the residues TYR34, TYR185, GLY187, ASP188, ILE189, SER299, PHE300, and THR303 within the active site of GPR52 were significant for efficient ligand binding through the formation of various hydrogen bond interactions and hydrophobic contacts. Out of the three hit compounds, compound 3 had the lowest ΔG of - 20.82 ± 0.44kcal/mol. The study identified compounds 1, 2, and 3 as potential molecules that can be developed as GPR52 inhibitors holding promise for lowering mHTT levels.

Phase 1b/2 Study of Escalating Doses of the NEDD8 Activating Enzyme Inhibitor Pevonedistat Administered in Combination with Standard Induction Therapy (Cytarabine and Idarubicin) in Newly Diagnosed High Risk Acute Myeloid Leukemia

Introduction: Pevonedistat is a potent and selective small-molecule inhibitor of the NEDD8-activating enzyme that has demonstrated clinical activity in acute myeloid leukemia (AML). Preclinical studies suggest that pevonedistat synergistically enhances the activity of cytarabine in AML. We investigated the safety and efficacy of pevonedistat in combination with 7+3 cytarabine and idarubicin induction chemotherapy in adults with high-risk AML. Methods: This open-label, multicenter phase 1b/2 study (NCT03330821) enrolled patients aged ≥18 years with newly diagnosed high-risk AML, defined as therapy-related AML, AML with antecedent myelodysplastic syndrome or AML with myelodysplasia-related changes, the presence of adverse genetic features per ELN 2017 guidelines, or being age ≥55. Eligible patients had an ECOG status of 0-2. Patients were administered pevonedistat on days 1, 3, and 5 of each 28-day cycle in combination with cytarabine 100 mg/m 2 on days 1-7 and idarubicin 12 mg/m 2 on days 1-3. In the phase 1b dose-escalation part of the study, a standard 3+3 design was used to evaluate 2 dose levels of pevonedistat: 15 and 20 mg/m 2. Consolidation consisted of up to 4 cycles of cytarabine (1-3 g/m 2 q12h) on days 1, 3, and 5 or transplant. For pharmacodynamic (PD) assessment, RT-PCR assays were performed on peripheral blood samples to measure expression levels of 7 NRF-2 target genes (ATF3, GCLM, GSR, NQ01, SLC7A11, SRXN1, and TXNRD1). The primary objective for the phase 1b study was to determine the RP2D of pevonedistat in combination with cytarabine and idarubicin. The primary objective for the phase 2 study was to determine the composite complete response rate (CRc; CR+CRi). Secondary objectives included relapse-free survival (RFS), overall survival (OS), and safety and tolerability. Results: A total of 28 patients were enrolled at 5 sites in the US between August 2018 and August 2021. Six patients were enrolled in the phase 1b study and 22 patients in phase 2. Baseline patient characteristics are shown in Table 1. The median age was 61 years (range 47-74). Per the 2017 ELN risk classification, 8 of 28 patients (29%) had favorable risk, 8 (29%) had intermediate risk, and 12 (43%) had adverse risk. All 28 patients were included for safety and response assessments. In phase 1b, none of the 6 patients experienced dose-limiting toxicity at 15 or 20 mg/m 2. Thus, the RP2D of pevonedistat was determined to be 20 mg/m 2. The CRc rate was 64% (95% CI 44-81) with 17 patients achieving CR and 1 patient achieving CRi. Of the 18 patients who achieved CR or CRi, 16 (89%) achieved MRD negativity by multiparameter flow cytometry, and 7 (39%) proceeded to HSCT. The CRc rates for patients in the ELN 2017 favorable, intermediate, and adverse risk groups were 100% (95% CI 63-100), 63% (95% CI 29-89), and 42% (95% CI 18-71), respectively. Eight patients were included in the interim PD analysis. Among patients who achieved CR or CRi (n=6), 4 (ATF3, GSR, SLC7A11, and SRXN1) of the 7 NRF-2 target genes measured were significantly upregulated at 5 hours post-pevonedistat induction compared to baseline. There was no significant increase in NRF-2-regulated gene transcript levels among patients with refractory disease (n=2). The median follow-up was 31.1 months (range 19.1-55.9). The median RFS was 24.4 months (95% CI 7-54.3+) and median OS was 25 months (95% CI 11.9-55.9+). The estimated 1-year and 2-year RFS were 72% (95% CI 52-93) and 55% (95% CI 23-78), respectively. The estimated 1-year and 2-year OS were 68% (95% CI 51-85) and 53% (95% CI 34-72), respectively. The most common grade ≥3 treatment-related adverse events were anemia (61%), febrile neutropenia (25%), and hypophosphatemia (18%). No treatment-related deaths were observed. Conclusion: Pevonedistat in combination with cytarabine and idarubicin was well tolerated among adults with newly diagnosed, high-risk AML and induced deep remissions with promising RFS and OS.

Discovery of BT-114143, a Novel and Potent Small-Molecule Plasminogen Inhibitor for Hyperfibrinolysis

Blood coagulation and fibrinolysis are essential physiological functions, whose dysregulation is a life-threatening emergent situation requiring immediate medical intervention. In particular, hyperfibrinolysis-triggered bleeding occurs in certain medical conditions such as postpartum hemorrhage (PPH) or during perioperative procedures, natural disasters, traumatic accidents as well as battlefield situations, and is one of the major causes of mortalities worldwide during peacetime and war. Tranexamic acid (TXA) has been widely used as an antifibrinolytic drug since 1960s, however, the low potency of TXA has limited its clinical applications. Here, we have developed BT-114143, a novel and potent small-molecule plasminogen inhibitor. BT-114143 exhibited over four-fold higher affinity for recombinant human plasminogen than TXA in surface plasmon resonance (SPR) analysis. Similarly, affinity of BT-114143 was at least one magnitude higher than that of either epsilon-aminocaproic acid (EACA) or p-aminomethylbenzoic acid (PAMBA) (Table 1). In vivo efficacy studies were conducted in recombinant tissue plasminogen activator (rt-PA)-triggered rat tail bleeding model and rabbit liver bleeding model by hepatectomy. BT-114143 reduced bleeding time dose-dependently in both animal models, but also exhibited similar potency as compared to TXA. Similar potency between BT-114143 and TXA was further confirmed by rt-PA triggered fibrinolysis in either rat or rabbit whole blood in thromboelastography (TEG) assay, suggesting interspecies difference in response to bleeding. However, ex vivo studies on human plasma clot lysis assay and human whole blood TEG assay (Figure 1) demonstrated nearly ten-fold antifibrinolytic potency as compared to TXA, suggesting superior efficacy for treatment of hyperfibrinolysis-related bleedings. In addition, a panel of GLP toxicology studies has confirmed the preclinical safety of BT-114143. Moreover, electrophysiological analysis has demonstrated comparable inhibitory effect of BT-114143 and TXA on GABA A receptor and Glycine receptor. At present, BT-114143 is undergoing phase I clinical study in China (CTR20222910) and is expected to apply for FDA approval for clinical trial in 2024. Furthermore, BT-114143 is being developed for intramuscular (i.m.) injection, making it more suitable for emergency situations including PPH, traumatic injuries, natural disasters, and combat injuries.

Zanubrutinib vs FCR in Fit Treatment-Naive Patients with Chronic Lymphocytic Leukemia: A Matching-Adjusted Indirect Comparison

Background: Fludarabine, cyclophosphamide, and rituximab (FCR) is the standard first-line therapy for fit (physically active, with no major health problems and normal renal function) treatment-naive patients with chronic lymphocytic leukemia (CLL) (Eichhorst et al. Ann Oncol. 2015). Due to the safety profile of FCR, including severe hematotoxicity, and severe infections, treatments with improved efficacy and safety are of interest (Hallek et al. Lancet. 2010; Fischer et al. Blood. 2016). Zanubrutinib, a highly specific and potent small-molecule inhibitor of Bruton tyrosine kinase, has been approved for treatment-naive patients with CLL. However, the comparative efficacy of zanubrutinib vs FCR in patients with CLL has not been investigated. Objective: The objective of this study was to determine the relative treatment effects of zanubrutinib vs FCR in fit treatment-naive patients with CLL. Methods: The CLL10 trial (NCT00769522; Eichhorst et al. Lancet Oncol. 2016) investigated FCR and bendamustine + rituximab (BR), while the SEQUOIA trial compared zanubrutinib to BR (NCT03336333; Tam et al. Lancet Oncol. 2022). Zanubrutinib and FCR were compared by means of an anchored matching-adjusted indirect comparison (MAIC) in which the BR arm of each trial served as a common comparator. Propensity score matching was conducted using patient-level data from SEQUOIA to adjust for differences between trial populations, per National Institute for Health and Care Excellence MAIC methods (Philippo et al. NICE DSU Technical Support Document 18. 2016). Progression-free survival (PFS), as assessed by the independent review committee, was then compared based on the matched patient populations. The selection of matching variables was based on literature review and consultation with clinical experts. As the indirect treatment comparison was anchored on the common comparator arm (BR), patient characteristics that are solely prognostic did not need to be incorporated. The core model included immunoglobulin heavy chain gene mutation, cytogenetic mutations (11q deletion), β2-microglobulin, Binet stage, and age. Geographic region, sex, creatinine clearance, Cumulative Illness Rating Scale (CIRS) score, Eastern Cooperative Oncology Group performance status (ECOG PS), and previous infections were tested in sensitivity analyses. Results: PFS was favorable with zanubrutinib vs FCR, and statistically significant differences were observed in the base case model (hazard ratio [HR], 0.41; 95% CI, 0.20-0.81; p-value=0.01). Sensitivity analyses showed significantly improved PFS with zanubrutinib when adding geographic region (HR, 0.43; 95% CI, 0.21-0.90; p-value=0.03), sex (HR, 0.44; 95% CI, 0.22-0.89; p-value=0.02), ECOG PS (HR, 0.30; 95% CI, 0.14-0.64; p-value<0.01), and previous infections (HR, 0.45; 95% CI, 0.22-0.93; p-value=0.03) to the base case set of matching variables. A sensitivity analysis incorporating CIRS (HR, 0.45; 95% CI, 0.16-1.24; p-value=0.12) showed only numerically favorable PFS with zanubrutinib, owing in part to the low effective sample size (ESS) in the expanded model. Similar results were obtained when creatinine clearance was added to the base case matching set of variables (HR, 0.52; 95% CI, 0.24-1.13; p-value=0.10). Conditional on the number of matching variables, ESS ranged from 174.1 in the core model to 64.05 in the analysis that included the CIRS score. Conclusion:Our findings suggest that zanubrutinib offers clinically meaningful benefits in PFS over FCR in fit treatment-naive patients with CLL. MAICs rely on reporting of relevant patient characteristics in published studies. In the current study, ZAP-70 methylation and TP53 mutation, which were considered treatment effect modifiers, were not reported in CLL10 and were not accounted for in the propensity model.