Potent Allosteric Inhibitor Research Articles

Proteomic Profiling of Serum Extracellular Vesicles Identifies Diagnostic Signatures and Therapeutic Targets in Breast Cancer.

Analysis of extracellular vesicles (EV) is a promising noninvasive liquid biopsy approach for breast cancer detection, prognosis, and therapeutic monitoring. A comprehensive understanding of the characteristics and proteomic composition of breast cancer-specific EVs from human samples is required to realize the potential of this strategy. In this study, we applied a mass spectrometry-based, data-independent acquisition proteomic approach to characterize human serum EVs derived from patients with breast cancer (n = 126) and healthy donors (n = 70) in a discovery cohort and validated the findings in five independent cohorts. Examination of the EV proteomes enabled the construction of specific EV protein classifiers for diagnosing breast cancer and distinguishing patients with metastatic disease. Of note, TALDO1 was found to be an EV biomarker of distant metastasis of breast cancer. In vitro and in vivo analysis confirmed the role of TALDO1 in stimulating breast cancer invasion and metastasis. Finally, high-throughput molecular docking and virtual screening of a library consisting of 271,380 small molecules identified a potent TALDO1 allosteric inhibitor, AO-022, which could inhibit breast cancer migration in vitro and tumor progression in vivo. Together, this work elucidates the proteomic alterations in the serum EVs of breast cancer patients to guide the development of improved diagnosis, monitoring, and treatment strategies. Significance: Characterization of the proteomic composition of circulating extracellar vesicles in breast cancer patients identifies signatures for diagnosing primary and metastatic tumors and reveals tumor-promoting cargo that can be targeted to improve outcomes.

140 (PB128): ATV-1601 is a Potent and Selective Allosteric Inhibitor of AKT1-E17K and Shows Profound and Durable Regressions in AKT1-E17K-Driven Patient Derived Xenograft Models

140 (PB128): ATV-1601 is a Potent and Selective Allosteric Inhibitor of AKT1-E17K and Shows Profound and Durable Regressions in AKT1-E17K-Driven Patient Derived Xenograft Models

In silico study of some plant compounds as potential anticancer agents targeting MALT1 allosteric domain

Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is the only human paracaspase, that serves as an adaptor protein and controls substantial genes expressed in the activation, proliferation of lymphocyte, and immune reactions by triggering the IKK/NF-kB signaling pathway. However, unusual MALT1-mediated NF-kB signaling pathway has been identified in multiple diseases like cancer, therefore making MALT1 a promising therapeutic target. There are scanty numbers of MALT1 inhibitors, thus the need to discover more compounds with less or no toxicity issue, that are cheap and pharmacologically efficient is of pertinence. Hence, our present study was to identify phyto-small molecules that could bind the allosteric interface of MALT1 using in silico methods. Total of 34 plant molecules were selected and screened for druglikeness, after which they were docked via Maestro 11.1 against the allosteric site of MALT1. The molecule with a binding score (kcal/mol) better than the control drug was subjected to molecular dynamics (MD) simulations of 100 ns via Desmond, free energy perturbations, principal component and Pearson correlation analyses. Our findings from this computational study presents cyanidin (–8.822 kcal/mol) as better binder to the allosteric site of MALT1 based on the molecular docking and pharmacokinetic profiling than thioridazine. Similarly, cyanidin-MALT1 complex showed significant stability and exhibiting contacts with critical amino acid residues in the site of interest than thioridazine-MALT1 complex. Hence, cyanidin is a potential allosteric inhibitor of MALT1. However, an urgent need for in vitro and in vivo validations is required to ascertain the efficacy of cyanidin in the fight against cancer and other MALT1-related diseases.

Discovery of JAB-3312, a Potent SHP2 Allosteric Inhibitor for Cancer Treatment.

As an oncogenic phosphatase, SHP2 acts as a converging node in the RTK-RAS-MAPK signaling pathway in cancer cells and suppresses antitumor immunity by passing signals downstream of PD-1. Here, we utilized the extra druggable pocket outside the previously identified SHP2 allosteric tunnel site by the (6,5 fused), 6 spirocyclic system. The optimized compound, JAB-3312, exhibited a SHP2 binding Kd of 0.37 nM, SHP2 enzymatic IC50 of 1.9 nM, KYSE-520 antiproliferative IC50 of 7.4 nM and p-ERK inhibitory IC50 of 0.23 nM. For JAB-3312, an oral dose of 1.0 mg/kg QD was sufficient to achieve 95% TGI in KYSE-520 xenograft model of mouse. JAB-3312 was well-tolerated in animal models, and a close correlation was observed between the plasma concentration of JAB-3312 and the p-ERK inhibition in tumors. Currently, JAB-3312 is undergoing clinical trials as a potential anticancer agent.

Design and synthesis of novel and potent allosteric HIV-1 integrase inhibitors with a spirocyclic moiety

We report herein the design and discovery of novel allosteric HIV-1 integrase inhibitors. Our design concept utilized the spirocyclic moiety to restrain the flexibility of the conformation of the lipophilic part of the inhibitor. Compound 5 showed antiviral activity by binding to the nuclear lens epithelium-derived growth factor (LEDGF/p75) binding site of HIV-1 integrase (IN). The introduction of a lipophilic amide substituent into the central benzene ring resulted in a significant increase in antiviral activity against HIV-1 WT X-ray crystallography of compound 15 in complex with the integrase revealed the presence of a hydrogen bond between the oxygen atom of the amide of compound 15 and the hydroxyl group of the T125 side chain. Chiral compound 17 showed high antiviral activity, good bioavailability, and low clearance in rats.

Discovery of azaleatin as a potential allosteric inhibitor for dengue NS2B-NS3 protease using in vitro and in silico studies

The rise in dengue cases in tropical and sub-tropical areas has become a significant health concern. At present, there is no definitive cure for dengue fever, which underscores the importance of identifying potent inhibitors. Dengue NS2B-NS3 protease is the prime drug target due to its vital function for replication. Quercetin, a flavone, has anti-dengue virus properties but is limited by low bioavailability. Previous studies have shown that methoxy substitution in flavones improves bioavailability and metabolic stability. Azaleatin is a derivative of quercetin with a methoxy substitution at the C5 position, however its ability to inhibit dengue is unknown. In this study, azaleatin was investigated for its inhibition against dengue NS2B-NS3 protease using in vitro and in silico techniques. The fluorescence assay was used to determine the IC50 value and inhibition kinetics. The molecular interaction between azaleatin and NS2B-NS3 was studied using CB-Dock2 and AutoDock Vina. The complex’s stability was then analysed using GROMACS. Besides, the ADMETlab 2.0 was utilized to predict pharmacokinetic of the azaleatin. Results showed that azaleatin inhibits dengue NS2B-NS3 protease non-competitively with a Ki of 26.82 µg/ml and an IC50 of 38 µg/ml. Molecular docking indicated binding of the azaleatin to the allosteric pocket of NS2B-NS3 with a docking score of −8.2 kcal/mol. Azaleatin was found stable in the pocket along 100 ns, supporting its inhibitory mode. The compound has favourable pharmacokinetic profiles and conformed to Lipinski’s Rule of Five. Taken together, azaleatin inhibits NS2B-NS3 protease in a non-competitive mode, suggesting its potential as safer anti-dengue compound. Communicated by Ramaswamy H. Sarma

Computational insights into allosteric inhibition of focal adhesion kinase: A combined pharmacophore modeling and molecular dynamics approach

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that modulates integrin and growth factor signaling pathways and is implicated in cancer cell migration, proliferation, and survival. Over the past decade various, FAK kinase, FERM, and FAT domain inhibitors have been reported and a few kinase domain inhibitors are under clinical consideration. However, few of them were identified as multikinase inhibitors. In kinase drug design selectivity is always a point of concern, to improve selectivity allosteric inhibitor development is the best choice. The current research utilized a pharmacophore modeling (PM) approach to identify novel allosteric inhibitors of FAK. The all-available allosteric inhibitor bound 3D structures with PDB ids 4EBV, 4EBW, and 4I4F were utilized for the pharmacophore modeling. The validated PM models were utilized to map a database of 770,550 compounds prepared from ZINC, EXIMED, SPECS, ASINEX, and InterBioScreen, aiming to identify potential allosteric inhibitors. The obtained compounds from screening step were forwarded to molecular docking (MD) for the prediction of binding orientation inside the allosteric site and the results were evaluated with the known FAK allosteric inhibitor (REF). Finally, 14 FAK-inhibitor complexes were selected from the docking study and were studied under molecular dynamics simulations (MDS) for 500 ns. The complexes were ranked according to binding free energy (BFE) and those demonstrated higher affinity for allosteric site of FAK than REF inhibitors were selected. The selected complexes were further analyzed for intermolecular interactions and finally, three potential allosteric inhibitor candidates for the inhibition of FAK protein were identified. We believe that identified scaffolds may help in drug development against FAK as an anticancer agent.

Inhibition of p38α MAPK-MK2 axis alleviate inflammatory response in various inflammation-related disease models

Abstract Mitogen-activated protein kinase (MAPK)-activated protein kinase-2 (MK2) is activated by p38 MAPK and regulates stability of mRNAs encoding inflammatory cytokines. Preclinical studies indicate that targeting MK2 may be a much safer approach than blocking p38a MAPK activation because that dissect the pathway downstream of p38α MAPK without affecting additional cellular functions. 141-A was identified as a highly potent and selective p38α MAPK-MK2 allosteric inhibitor that exhibited nanomolar-level inhibition on p38α MAPK activation of MK2 while sparing the p38α MAPK activation of ATF2 and PRAK. 141-A potently reduced various cytokine production (TNFα, IL-1β, IL-6, IL-8 and IL-17) in human peripheral blood mononuclear cells (PBMC) and displayed excellent efficacy in adjuvant-induced arthritis (AIA), psoriasis and MSU-induced air pouch rat models with 1/6 of the systematic exposure of CDD450. The compound exhibits favorable physicochemical and pharmacokinetic properties, along with an excellent preclinical toxicity profile, which may enhance therapeutic efficacy without causing side effects. 141-A is currently under IND-enabling studies and P1 trial is planned in early 2025.

Abstract 1975: Discovery of I-1000233, a potent, selective and brain permeable SHP2 allosteric inhibitor

Abstract Background: SHP2 (Src homology region 2-containing protein tyrosine phosphatase 2) is a target of interest for cancer therapy due to its key role in the regulation of RAS/MAPK signal transduction downstream of Receptor Tyrosine Kinases (RTKs). We report here the identification of I-1000233, a novel, highly potent, orally available and brain permeable SHP2 allosteric inhibitor, with potential for the treatment of tumors with dysregulated RTK/RAS/ERK signaling pathways. Material and Methods: High-throughput biological, biochemical and pharmacodynamic (PD) assays were used to inform Structure Activity Relationship studies which led to the identification of a chemical series of potent SHP2 inhibitors. Iterative optimization of physicochemical and pharmacological properties resulted in the identification of I-1000233, an orally available SHP2 inhibitor that showed excellent drug-like properties in preclinical studies. Results: I-1000233 is a potent inhibitor of the SHP2 enzyme in vitro (IC50 = 2 nM) with an exceptionally long residence time on the target. When tested in cell lines, it exhibits significant anti-proliferation activity against brain tumor cells and multiple RAS or EGFR mutant models. It strongly inhibits pERK, a downstream marker of MAPK pathway activity, in a dose dependent manner. Further, in vivo preclinical data confirmed that I-1000233 penetrates the BBB and shows anti-tumor activity, supporting its potential in the treatment of CNS and brain-metastasized tumors. Conclusions: I-1000233 is a potent, selective and orally available SHP2 inhibitor with potential for once-a-day dosing and its brain permeability in humans makes it a promising therapeutic agent against SHP2i responsive brain tumors and metastases. Citation Format: Francesca Puca, Danilo Fabbrini, Alina Ciammaichella, Antonino Missineo, Monica Bisbocci, Martina Nibbio, Annalise Di Marco, Claudia Apicella, Fabrizio Colaceci, Romina Sasso, Daniela Natale, Francesco Scalabrì, Cristina Alli, Vincenzo Pucci, Christian Montalbetti, Alessia Petrocchi, Alessandro Carugo, Carlo Toniatti. Discovery of I-1000233, a potent, selective and brain permeable SHP2 allosteric inhibitor [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 1975.

Abstract 6586: ORIC-944, a potent and selective allosteric PRC2 inhibitor with best-in-class properties, demonstrates combination synergy with AR pathway inhibitors in prostate cancer models

Abstract Polycomb repressive complex 2 (PRC2) dysregulation occurs in multiple tumor types and is associated with poor prognosis in patients with prostate cancer. In patients treated with androgen receptor pathway inhibitors (ARPI), emergence of tumor cell plasticity is associated with resistance to therapy. Dysregulation of epigenetic reprogramming factors, including PRC2, creates an environment that is permissive for such lineage plasticity. Inhibition of the PRC2 complex therefore may provide an opportunity to overcome or prevent the emergence of plasticity in prostate cancer. Notably, emerging clinical trial data has demonstrated the potential of ARPI and PRC2 inhibitor combination therapy to improve outcomes in patients with metastatic prostate cancer. PRC2 tri-methylates histone H3 at lysine 27 (H3K27me3), leading to long-term transcriptional silencing, a key mechanism regulating cellular functions such as cell growth and differentiation. Three core subunits comprise PRC2: the catalytic subunit enhancer of zeste homolog 2 (EZH2), suppressor of zeste 12 (SUZ12) and embryonic ectoderm development (EED). EED is essential for the histone methyltransferase activity of PRC2. First-generation PRC2 inhibitors, which target EZH2, exhibit poor drug properties, drug-drug interaction liabilities and short half-life requiring twice daily dosing at high doses in patients. We developed a second generation PRC2 inhibitor, ORIC-944, an allosteric inhibitor of PRC2 that binds the EED subunit. ORIC-944 is a potent, highly selective, orally bioavailable inhibitor with best-in-class properties. ORIC-944 demonstrated single agent tumor growth inhibition in a spectrum of in vivo prostate cancer models, including AR-positive, AR-mutant, ARv7, ARPI-responsive and ARPI-resistant models. In vitro analysis of the combination of ARPIs and ORIC-944 showed synergy in multiple prostate cancer cells, utilizing BLISS and other quantitative methods. In vivo studies revealed that ORIC-944 demonstrated increased efficacy in combination with ARPI, consistent with the in vitro synergy. RNA-seq analysis of transcriptional changes induced by ORIC-944 provided mechanistic insight into the role of PRC2 in prostate cancer lineage plasticity and combination response. These results position ORIC-944 as a best-in-class PRC2 inhibitor for evaluation in combination with ARPI in patients with metastatic prostate cancer. A phase 1b trial of ORIC-944 is ongoing (NCT05413421). Citation Format: Anneleen Daemen, Natalie Yuen, Amber Wang, Aleksandr Pankov, Livia Ulicna, Chelsea Chen, Frank L. Duong, Jason Long, Matthew A. Marx, James G. Christensen, Lori S. Friedman, Melissa R. Junttila. ORIC-944, a potent and selective allosteric PRC2 inhibitor with best-in-class properties, demonstrates combination synergy with AR pathway inhibitors in prostate cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6586.

Abstract 7583: Discovery of a potent small molecule MALT1 protease inhibitor for the treatment of cancer

Abstract The paracaspase mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1), as the core component of CBM complex (CARD9/10/11/14-BCL10-MALT1), plays an essential role in activating the NF-κB pathway and regulating the cell proliferation, differentiation and immunity. Inhibiting of MALT1 is considered a promising therapeutic approach for the treatment of several non-Hodgkin B-cell lymphomas, chronic lymphocytic leukemia (CLL), solid tumors and autoimmune diseases. Herein, we report the discovery of a novel potent MALT1 allosteric inhibitor, BPI-530616, which strongly inhibited MALT1 protease activity with subnanomolar IC50, and low off-target activity on other proteases. BPI-530616 can inhibit the proliferation of B-cell diffuse large B-cell lymphoma (ABC-DLBCL) cell line, OCI-ly3, bearing CD79b and CARD11 mutations. The cell line is intrinsically resistant to covalent and non-covalent Bruton's Tyrosine Kinase (BTK) inhibitors. Meanwhile, BPI-530616 suppressed the cellular cytokine secretion (e.g.IL-10, IL-2) or MALT1 direct substrate cleavage (e.g. CYLD). In vivo, BPI-530616 dose-dependently suppressed tumor growth and inhibited the cleavage of CYLD in OCI-ly3 xenograft mouse model. To address the role of MALT1 inhibitor in the contest of immune-oncology, a CT26 syngeneic mouse model was set up. Oral administration of BPI-530616 exhibited a good antitumor effect comparable to that of the anti-PD1 antibody, with no observable adverse effects. Additionally, BPI-530616 displayed desirable drug-like properties, including low clearance and good oral bioavailability across multiple pre-clinical species. Taken together, our research has led to the discovery of a novel, potent, selective, and orally bioavailable small molecule MALT1 inhibitor, BPI-530616. It demonstrated anti-tumor efficacy in the DLBCL animal model and showed promising effects in an immune-oncology animal model. Citation Format: Yuzhen Zhou, Bang Fu, Jie Chen, Weidong Cai, Xiao Sun, Yinlong Li, Wei Ren, Qichao Shen, Guolong Du, Xiaojun Zhou, Zhengyao Zou, Haibo Chen, Xiaoyun Liu, Cheng Yang, Xiangyong Liu, Jing Guo, Hao Wu, Lieming Ding, Jiabing Wang, Hong Lan. Discovery of a potent small molecule MALT1 protease inhibitor for the treatment of 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 7583.

Discovery of N-benzylbenzamide-based allosteric inhibitors of Aurora kinase A

Aurora kinases (AurkA/B/C) regulate the assembly of bipolar mitotic spindles and the fidelity of chromosome segregation during mitosis, and are attractive therapeutic targets for cancers. Numerous ATP-competitive AurkA inhibitors have been developed as potential anti-cancer agents. Recently, a few allosteric inhibitors have been reported that bind to the allosteric Y-pocket within AurkA kinase domain and disrupt the interaction between AurkA and its activator TPX2. Herein we report a novel allosteric AurkA inhibitor (6h) of N-benzylbenzamide backbone. Compound 6h suppressed the both catalytic activity and non-catalytic functions of AurkA. The inhibitory activity of 6h against AurkA (IC50 = 6.50 μM) was comparable to that of the most potent allosteric AurkA inhibitor AurkinA. Docking analysis against the Y-pocket revealed important pharmacophores and interactions that were coherent with structure–activity relationship. In addition, 6h suppressed DNA replication in G1-S phase, which is a feature of allosteric inhibition of AurA. Our current study may provide a useful insight in designing potent allosteric AurkA inhibitors.

Binding Free Energy Calculation Based on the Fragment Molecular Orbital Method and Its Application in Designing Novel SHP-2 Allosteric Inhibitors.

The accurate prediction of binding free energy is a major challenge in structure-based drug design. Quantum mechanics (QM)-based approaches show promising potential in predicting ligand-protein binding affinity by accurately describing the behavior and structure of electrons. However, traditional QM calculations face computational limitations, hindering their practical application in drug design. Nevertheless, the fragment molecular orbital (FMO) method has gained widespread application in drug design due to its ability to reduce computational costs and achieve efficient ab initio QM calculations. Although the FMO method has demonstrated its reliability in calculating the gas phase potential energy, the binding of proteins and ligands also involves other contributing energy terms, such as solvent effects, the 'deformation energy' of a ligand's bioactive conformations, and entropy. Particularly in cases involving ionized fragments, the calculation of solvation free energy becomes particularly crucial. We conducted an evaluation of some previously reported implicit solvent methods on the same data set to assess their potential for improving the performance of the FMO method. Herein, we develop a new QM-based binding free energy calculation method called FMOScore, which enhances the performance of the FMO method. The FMOScore method incorporates linear fitting of various terms, including gas-phase potential energy, deformation energy, and solvation free energy. Compared to other widely used traditional prediction methods such as FEP+, MM/PBSA, MM/GBSA, and Autodock vina, FMOScore showed good performance in prediction accuracies. By constructing a retrospective case study, it was observed that incorporating calculations for solvation free energy and deformation energy can further enhance the precision of FMO predictions for binding affinity. Furthermore, using FMOScore-guided lead optimization against Src homology-2-containing protein tyrosine phosphatase 2 (SHP-2), we discovered a novel and potent allosteric SHP-2 inhibitor (compound 8).

Study of Gnetum gnemon metabolites as potential anti-breast and prostate cancer by metabolomic and molecular docking

Breast and prostate cancers are significant health concerns worldwide, demanding novel and effective therapeutic approaches. This study explores the potential of Gnetum gnemon seed metabolites as anticancers agent through metabolomic profiling and molecular docking analysis. Six metabolites from G. gnemon seed were identified, which met Lipinski’s rule of five criteria, suggesting their drug-like properties. A panel of 10 molecular target proteins, including PTGS1, PTGS2, ESR1, SIRT1, SIRT3, SIRT5, AKT1, JAK2, BRAF, and NOS3, relevant to breast and prostate cancer pathways, were selected for molecular docking simulations. The binding interactions and free binding energy assessments identified gnetol and gnetin C as the most promising metabolites, showing strong interactions with multiple target proteins. Gnetol exhibited potential in targeting ESR1 and SIRT5, suggesting a mechanism for breast cancer inhibition. On the other hand, gnetin C emerged as a potent allosteric inhibitor of AKT1, potentially impacting breast and prostate cancer pathways. These findings highlight G. gnemon metabolites, particularly gnetol and gnetin C, as potential candidates for further preclinical and clinical studies as anti-breast and prostate cancer agents.

A potential allosteric inhibitor of SARS-CoV-2 main protease (Mpro) identified through metastable state analysis.

Anti-COVID19 drugs, such as nirmatrelvir, have been developed targeting the SARS-CoV-2 main protease, Mpro, based on the critical requirement of its proteolytic processing of the viral polyproteins into functional proteins essential for viral replication. However, the emergence of SARS-CoV-2 variants with Mpro mutations has raised the possibility of developing resistance against these drugs, likely due to therapeutic targeting of the Mpro catalytic site. An alternative to these drugs is the development of drugs that target an allosteric site distant from the catalytic site in the protein that may reduce the chance of the emergence of resistant mutants. Here, we combine computational analysis with in vitro assay and report the discovery of a potential allosteric site and an allosteric inhibitor of SARS-CoV-2 Mpro. Specifically, we identified an Mpro metastable state with a deformed catalytic site harboring potential allosteric sites, raising the possibility that stabilization of this metastable state through ligand binding can lead to the inhibition of Mpro activity. We then performed a computational screening of a library (∼4.2 million) of drug-like compounds from the ZINC database and identified several candidate molecules with high predicted binding affinity. MD simulations showed stable binding of the three top-ranking compounds to the putative allosteric sites in the protein. Finally, we tested the three compounds in vitro using a BRET-based Mpro biosensor and found that one of the compounds (ZINC4497834) inhibited the Mpro activity. We envisage that the identification of a potential allosteric inhibitor of Mpro will aid in developing improved anti-COVID-19 therapy.

Design and synthesis of (E)-3-benzylideneindolin-2-one derivatives as potential allosteric inhibitors of Aurora A kinase

The mitotic kinase Aurora A, a pivotal regulator of cell cycle, is overexpressed in various cancers and has emerged as one of the most promising targets for anticancer drug discovery....

Integrating machine learning and high throughput screening for the discovery of allosteric AKT1 inhibitors

Evidence from clinical and experimental investigations reveals the role of AKT in oral cancer, which has led to the development of therapeutic and pharmacological medications for inhibiting AKT protein. Despite prodigious effort, researchers are searching for new allosteric inhibitors as orthosteric inhibitors are non-selective and exert off-target effects. In the current study, we proposed an integrated computational workflow for identifying allosteric AKT1 inhibitors as this isoform is highly correlated with poor prognosis and survival. To achieve this objective, 84 classification QSAR models with six different machine learning algorithms were developed. The models created with RDKit_RF and RDKit_kstar outperformed internal and test set validation with an ROC of 0.98. The outperformed models were then used to screen Chembl, which contains over a million compounds, for AKT1 inhibitors. The Tanimoto similarity search approach identified the compounds structurally resembling AKT allosteric inhibitors. The filtered compounds were further subjected to docking phases, molecular dynamic simulation and mmpbsa to verify the binding mode of selected ones. All these analyses suggested hit 5 (CHEMBL3948083) as the potential allosteric inhibitor of AKT1 as the stability parameters, favourable binding affinity (-107.78 ± 11.56 KJ/mol) and ligand interaction were better in comparison to other compounds and reference compound. The residual analysis demonstrated that allosteric and isoform-specific residues such as Trp80 and Val270 contributed the larger energy for ligand binding. The proposed integrated approach in this study might achieve a futuristic outcome when employed in a pharmaceutical scheme different from the conventional method. Communicated by Ramaswamy H. Sarma

Abstract C075: A first-in-class and highly selective SHP1 allosteric inhibitor exhibits robust anti-tumor immunity and synergizes with PD-1 blockade

Abstract Background: SHP1 is a member of the src homology 2 (SH2) domain-containing protein phosphatases. It contains two SH2 domains at the N-terminus, a catalytic domain and a C-terminal tail. SHP1 is primarily expressed in hematopoietic cells and commonly recognized as a negative regulator of multiple signaling pathways. It has been known that SHP1 interacts with proteins of the inhibitory-receptor superfamily containing immunoreceptor tyrosine-based inhibitory motifs. Methods: To screen for SHP1 allosteric inhibitors, we employed a DiFMUP high-throughput biochemical assay system. Compounds that only perturbed the activity of SHP1 was further optimized for cellular activity on target, in vitro ADME, and in vivo pharmacokinetic properties. X-ray crystallography has been introduced to determine the interaction between the inhibitor and the allosteric site of SHP1. We also evaluated the lead series in in-vitro studies with human primary immune cells and in-vivo efficacy studies in syngeneic mouse models. Results: We discovered a series of SHP1 allosteric inhibitors with IC50 values at the nanomolar level and high selectivity over the other PTP enzymes, including SHP2. Structurally, SHP1 adopts an autoinhibited conformation in its basal state, where the N-SH2 domain interacts with the PTP domain and blocks access to the catalytic site. X-ray crystallography reveals that our SHP1 inhibitors are bound to non-catalytic sites of the SHP1 inactive conformation. Among the compounds, SB6299 demonstrated potent cytokine responses in various human primary immune cell types. SB6299 significantly enhanced IL-2, IFN-γ, and TNF-α productions in human primary pan-T, NK, and monocyte cells, respectively. The cytokine release was confirmed as a result of selective SHP1 inhibition through the evaluation of SHP1 knockout in primary immune cells. Moreover, SB6299 showed dose responses in costimulatory molecules and cytokines (TNF-α and IL6) in human monocyte derived dendritic cells (DCs) and enhanced DC maturation markers such as CD80, CD86, and CD40. The anti-tumor efficacy of SB6299 was evaluated in the CT26 and MC38 syngeneic mouse models. Orally administered SB6299 showed tumor growth inhibition as a single agent, and the efficacy was further enhanced when administered in combination with an anti-PD1 antibody. Conclusions: 1. Our studies successfully demonstrate that SHP1 can be allosterically inhibited, allowing selectivity with other phosphatases, including SHP2. 2. SB6299 is the first-in-class orally available, potent, and selective SHP1 allosteric inhibitor. 3. Attenuation of SHP1 activity by SB6299 could promote cytokine secretion and cytotoxic effects in human primary immune cells. 4. Orally administered SB6299 induced tumor growth inhibition as a single agent and in combination with an anti-PD1 antibody. 5. Currently, more selective and potent inhibitors are being tested. Citation Format: Jun Gyu Kim, Ki Moon Ryu, Mi Yeon Jang, Jongho Cho, Dajeong Kim, Dae Hyeon Seong, Kyu Hwan Kim, Ik-Soo Jang, Jeong Yoon Shin, Hong Sik Han, Joon Yonug Hwang, Dae Young Lee, Chae Lim Ryu, Song Yi Lee, Tae-Dong Han, SeungMin Yang, Hyounmie Doh. A first-in-class and highly selective SHP1 allosteric inhibitor exhibits robust anti-tumor immunity and synergizes with PD-1 blockade [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 C075.

Sgr-1505 Is a Potent MALT1 Protease Inhibitor with a Potential Best-in-Class Profile

Background: MALT1 (Mucosa-associated lymphoid tissue lymphoma translocation protein 1) is a component of the MALT1-BCL10-CARD11 complex downstream from the Bruton Tyrosine Kinase (BTK) on the B-cell receptor signaling pathway. MALT1 is a key mediator of nuclear factor kappa B (NF-κB) signaling, which is the main driver of a subset of B-cell lymphomas. MALT1 is considered a potential therapeutic target for several subtypes of non-Hodgkin B-cell lymphomas and chronic lymphocytic leukemia (CLL), including tumors with acquired BTK inhibitor (BTKi) resistance. Constitutive activation of the NF-κB is a molecular hallmark of activated B cell-like diffuse large B cell lymphoma (ABC-DLBCL), and MALT1 may have utility as a treatment option for ABC-DLBCL. Previously, we described the discovery of novel MALT1 inhibitors with anti-proliferative effects in non-Hodgkin B-cell lymphoma cells and the strong anti-tumor activity of our MALT1 inhibitors across multiple tumor models as well as combination potential with agents including standard-of-care (ref 1, 2). SGR-1505 is an oral potent small molecule allosteric inhibitor of MALT1 that inhibits MALT1 enzymatic activity and demonstrates anti-proliferative activity in ABC-DLBCL cell lines, both BTKi-sensitive (OCI-LY10) and BTKi-resistant (OCI-LY3). When administered as a single agent and in combination with the approved Bruton's tyrosine kinase (BTK) inhibitor, ibrutinib, SGR-1505 demonstrated tumorostatic and regressive antitumor activity in ABC-DLBCL cell line-derived xenograft and patient-derived xenograft models. These data suggest that SGR-1505-mediated MALT1 inhibition has therapeutic potential for patients with selected B-cell lymphomas. Here we further characterized SGR-1505, in a series of in vitro and ex vivo assays, as well as RNA-seq analysis to examine changes in gene expression from in vivo tumor samples. We also compared SGR-1505 with a competitor Phase I candidate, JNJ-67856633 (JNJ-6633) (ref 3, 4). Results: SGR1505 potency and downstream effects were evaluated in a series of biochemical and cell based assays. SGR-1505 showed excellent potency in the biochemical assay and strong anti-proliferative effects on ABC-DLBCL cells. SGR-1505 was more potent than JNJ-6633 in all assays tested (Table 1). These results are also consistent with the result from a human primary T-cell based assay, where SGR-1505 showed at least ten-fold better potency than JNJ-6633. RNA-seq analysis was conducted to examine changes in gene expression from in vivo tumor samples. Greater modulation of BIOCARTA NF-kB pathway genes was seen with SGR-1505 compared to JNJ-6633, as measured by mean absolute change in gene expression. At 6 hr and later timepoints, we also observed a trend of increases in genes related to cell cycle pathways, such as cell cycle, DNA damage, and apoptosis. SGR-1505 is being evaluated in the SGR-1505-102 phase 1 study, which is an ongoing first-in-human, single center, dose escalation study to evaluate the safety, tolerability, PK and PD of SGR-1505 tablets in healthy participants (ACTRN12623000358640p). Preliminary data showed changes in target engagement markers at concentrations predicted by the in vitro and ex vivo assays, consistent with MALT1 protease inhibition. Conclusions: SGR-1505, a MALT1 protease small molecule inhibitor, consistently demonstrated better potency in in vitro and ex vivo assays when compared to the clinical-stage JNJ-6633 compound and greater effects on NF-kB pathway gene expression in in vivo tumor samples based on RNA-seq analysis. Changes in biological pathways mediated by MALT1 were also observed at relevant doses in the ongoing SGR-1505 healthy volunteer study. Currently, a phase 1 clinical trial in patients with mature B cell neoplasms is also ongoing (NCT05544019). The data presented suggests SGR-1505 has a potential best-in-class profile and supports advancing the ongoing clinical development of SGR-1505.

A Phase 1, Open-Label, Multicenter, Dose-Escalation Study of Sgr-1505 As Monotherapy in Subjects with Mature B-Cell Malignancies

Background and Significance: MALT1 (Mucosa-associated lymphoid tissue lymphoma translocation protein 1) is a component of the MALT1-BCL10-CARD11 complex downstream from the Bruton Tyrosine Kinase (BTK) on the B-cell receptor signaling pathway. MALT1 is a key mediator of nuclear factor kappa B (NF-κB) signaling, which is the main driver of a subset of B-cell lymphomas. MALT1 is considered a potential therapeutic target for several subtypes of non-Hodgkin B-cell lymphomas and chronic lymphocytic leukemia (CLL), including tumors with acquired BTK inhibitor (BTKi) resistance. Constitutive activation of NF-κB is a molecular hallmark of activated B cell-like diffuse large B cell lymphoma (ABC-DLBCL), and MALT1 may have utility as a treatment option for ABC-DLBCL. Furthermore, a MALT1 inhibitor (JNJ-67856633) showed efficacy in mature B cell malignancies from phase 1 studies (ref 1, 2). SGR-1505 is an oral potent small molecule allosteric inhibitor of MALT1 that inhibits MALT1 enzymatic activity and demonstrates anti-proliferative activity in BTKi-sensitive (OCI-LY10) and BTKi-resistant (OCI-LY3) ABC-DLBCL cell lines. SGR-1505 administered as a single agent and in combination with the approved Bruton's tyrosine kinase (BTK) inhibitor, ibrutinib, demonstrates tumorostatic and regressive antitumor activity in ABC-DLBCL cell line-derived and patient-derived xenograft models. These data suggest that SGR-1505-mediated MALT1 inhibition may expand therapeutic options for patients with selected B-cell lymphomas, supporting further evaluation of SGR-1505 in clinical trials. Study Design and Methods: SGR-1505-101 (NCT05544019) is a phase 1, multicenter trial of SGR-1505 as monotherapy in subjects with mature B-cell malignancies (figure 1). At present, the study is open to accrual at multiple investigative sites in the US with plans to expand to Europe. The primary objective is to evaluate safety and tolerability of SGR-1505 as monotherapy and to identify the maximum tolerated dose (MTD) and/or recommended dose (RD). Secondary objectives are to evaluate the pharmacokinetic (PK) profile, food effect, drug-drug interaction, and preliminary anti-tumor activity of SGR-1505. Key inclusion criteria are: history of mature B-cell malignancy (including aggressive and indolent B-cell lymphomas, Waldenström macroglobulinemia, and CLL); measurable or detectable disease according to the applicable disease-specific classification system (Lugano, iwCLL, WWM6); Eastern Cooperative Oncology Group (ECOG) performance status of ≤ 2. Patients with indolent B-cell lymphomas or CLL must have an indication for treatment and not require immediate cytoreductive therapy. Subjects with symptomatic or active CNS involvement, and other conditions or laboratory findings placing them at increased risk to the use of an investigational drug are excluded. SGR-1505 is initially dose-escalated using an accelerated titration design in cohorts of 1-6 subjects, and at higher dose levels using a conventional 3+3 design.