Small Molecule Allosteric Inhibitors Research Articles

Native dynamics and allosteric responses in PTP1B probed by high-resolution HDX-MS.

Protein tyrosine phosphatase 1B (PTP1B) is a validated therapeutic target for obesity, diabetes, and certain types of cancer. In particular, allosteric inhibitors hold potential for therapeutic use, but an incomplete understanding of conformational dynamics and allostery in this protein has hindered their development. Here, we interrogate solution dynamics and allosteric responses in PTP1B using high-resolution hydrogen-deuterium exchange mass spectrometry (HDX-MS), an emerging and powerful biophysical technique. Using HDX-MS, we obtain a detailed map of backbone amide exchange that serves as a proxy for the solution dynamics of apo PTP1B, revealing several flexible loops interspersed among more constrained and rigid regions within the protein structure, as well as local regions that exchange faster than expected from their secondary structure and solvent accessibility. We demonstrate that our HDX rate data obtained in solution adds value to estimates of conformational heterogeneity derived from a pseudo-ensemble constructed from ~200 crystal structures of PTP1B. Furthermore, we report HDX-MS maps for PTP1B with active-site versus allosteric small-molecule inhibitors. These maps suggest distinct and widespread effects on protein dynamics relative to the apo form, including changes in locations distal (>35 Å) from the respective ligand binding sites. These results illuminate that allosteric inhibitors of PTP1B can induce unexpected changes in dynamics that extend beyond the previously understood allosteric network. Together, our data suggest a model of BB3 allostery in PTP1B that combines conformational restriction of active-site residues with compensatory liberation of distal residues that aid in entropic balancing. Overall, our work showcases the potential of HDX-MS for elucidating aspects of protein conformational dynamics and allosteric effects of small-molecule ligands and highlights the potential of integrating HDX-MS alongside other complementary methods, such as room-temperature X-ray crystallography, NMR spectroscopy, and molecular dynamics simulations, to guide the development of new therapeutics.

Small Molecule Allosteric Inhibitor of HIPK2 as a Novel Therapy against Kidney Fibrosis.

Small Molecule Allosteric Inhibitor of HIPK2 as a Novel Therapy against Kidney Fibrosis.

Identification of new small molecule allosteric SHP2 inhibitor through pharmacophore-based virtual screening, molecular docking, molecular dynamics simulation studies, synthesis and in vitro evaluation

Src homology-2 (SH2) domain-containing phosphatase-2 (SHP2) is the first identified protooncogene and is a promising target for developing small molecule inhibitors as cancer chemotherapeutic agents. Pharmacophore-based virtual screening (PBVS) is a pharmacoinformatics methodology that employs physicochemical knowhow of the chemical space into the dynamic environs of computational technology to extract virtual molecular hits that are precise and promising for a drug target. In the current study, PBVS has been applied on EnamineTM Advanced Collection of 551,907 molecules by using a pharmacophore model developed upon SHP099 by Molecular Operating Environment (MOE) software to identify potential small molecule allosteric SHP2 inhibitors. Obtained 37 hits were further filtered through DruLiTo software for drug-likeness and PAINS remover which yielded 35 hits. These were subjected to molecular docking studies against the tunnel allosteric site of SHP2 (PDB ID: 5EHR) to screen them according to their binding affinity for the enzyme. Top 5 molecules having highest binding affinity for 5EHR were passed through an ADMET prediction screening and the top 2 hits (ligands 111675 and 546656) with the most favourable ADMET profile were taken for post screening molecular docking and MD simulation studies. From the protein-ligand interaction pattern, conformational stability and energy parameters, ligand 111675 (SHP2 Ki = 0.118 µM) resulted as the most active molecule. Further, the synthesis and in vitro evaluation of the lead compound 111675 unveiled its potent inhibitory activity (IC50 = 0.878 ± 0.008 µM) against SHP2. Communicated by Ramaswamy H. Sarma

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.

Safety, tolerability, pharmacokinetics, and pharmacodynamics of single and multiple doses of aficamten in healthy Chinese participants: a randomized, double-blind, placebo-controlled, phase 1 study.

Objectives: Aficamten is a selective, small-molecule allosteric inhibitor of cardiac sarcomere being developed as a chronic oral treatment for patients with symptomatic obstructive hypertrophic cardiomyopathy. This was the first-in-Chinese study aiming to investigate the safety, tolerability, pharmacokinetics, and pharmacodynamics of aficamten in healthy adults. Methods: This double-blind, randomized, placebo-controlled, phase 1 study was conducted in 28 healthy male and female Chinese participants after single ascending dose (SAD) and multi-dose (MD) administrations of aficamten. In the SAD cohort, 16 participants were randomized to receive a single oral dose of aficamten: 10mg, 20mg, or placebo. In the MD cohort, 12 participants were randomized to receive multiple doses of aficamten: 5mg or placebo once daily for 14days. Safety was monitored throughout the study with electrocardiograms, echocardiograms, clinical laboratory tests, and reporting of adverse events (AEs). Pharmacokinetic profiles of aficamten and metabolites, as well as CYP2D6 genetic impact, were evaluated. Results: A total of 35 treatment-emergent AEs were reported by 14 (50%) participants with mild severity. There were no serious AEs or adverse decreases in left ventricular ejection fraction below 50% during the study. Aficamten was dose-proportional over the dose range of 5-20mg and accumulated in the MD cohort. Conclusion: Aficamten was safe and well-tolerated in the healthy Chinese adult participants. The pharmacokinetics of aficamten in the Chinese population was comparable to those previously found in Western participants. These phase 1 data support the progression of aficamten into future clinical studies in Chinese patients. Clinical Trial registration: https://clinicaltrials.gov, identifier: NCT04783766.

A phase 1, multicenter, open-label, dose-escalation and dose-expansion study to evaluate the safety, tolerability, pharmacokinetics (PK), and efficacy of HS-10382 (TERN-701) in patients (pts) with chronic myeloid leukemia (CML).

TPS7081 Background: There is a need for new treatment options for pts with CML who are intolerant/resistant to currently available BCR-ABL1 ATP-binding site targeted tyrosine kinase inhibitors (TKIs). HS-10382 (TERN-701) is a potent, oral small molecule allosteric inhibitor of BCR-ABL. By binding to the myristoyl site of the BCR-ABL1 protein, HS-10382 (TERN-701) locks BCR-ABL1 into an inactive conformation. This results in a mutation-driven resistance profile different from that of ATP-binding site TKIs, providing potential for both monotherapy and combination therapy with ATP-binding site TKIs. In preliminary nonclinical assays, HS-10382 (TERN-701) was highly potent against wild type BCR-ABL1 as well as most common mutations acquired by pts treated with second generation active site TKIs. This ongoing first-in-human phase 1 study is evaluating HS-10382 (TERN-701) in pts with CML. Methods: Eligible adult CML chronic phase (CP) or advanced phase (AP) pts who are resistant or intolerant to prior BCR-ABL-1 TKI therapy are enrolled in an open-label Phase 1 study to evaluate the safety, tolerability, PK and efficacy of HS-10382 (TERN-701). This study is being conducted in two parts: Part 1 dose escalation utilizing a rolling six design and Part 2 dose expansion. In the dose escalation stage, CML-CP/AP pts who are resistant or intolerant to prior BCR-ABL-1 therapy are being enrolled to determine the maximum tolerated dose (MTD) or maximum applicable dose. Doses selected from the escalation part will be evaluated in the expansion stage in CML-CP pts who are resistant or intolerant to prior BCR-ABL-1 TKI therapy and have no T315I mutation. All pts will be carefully followed for adverse events for the duration of study treatment and for 28 days after the last dose of study drug. Pts will be permitted to continue therapy with assessments for progression if the study drug is well-tolerated and the pt has no objective evidence of disease progression. This study is ongoing (enrollment underway for Part 1, dose 3 as of February 2023) with 7 participating study sites in China. Clinical trial information: NCT05367700 .

SGR‐1505‐101: A PHASE 1, OPEN‐LABEL, MULTICENTER, DOSE‐ESCALATION STUDY OF SGR‐1505 AS MONOTHERAPY IN SUBJECTS WITH MATURE B‐CELL MALIGNANCIES

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 the 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. In particular, 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. 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). 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 xenograft and patient-derived xenograft models. Aims: These data suggest that SGR-1505-mediated MALT1 inhibition has therapeutic potential and may expand therapeutic options for patients with selected B-cell lymphomas supporting further evaluation of SGR-1505 in clinical trials. Methods: SGR-1505-101 (NCT05544019) is a phase 1, multicenter trial of SGR-1505 as monotherapy in subjects with mature B-cell malignancies. At present, the study has been activated in 3 sites in the United States. 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. Encore Abstract—previously submitted to EHA 2023 The research was funded by: Schrodinger Keywords: aggressive B-cell non-Hodgkin lymphoma, molecular targeted therapies, ongoing trials Conflicts of interests pertinent to the abstract. B. Yoo Employment or leadership position: Schrodinger Stock ownership: Schrodinger Z. Nie Employment or leadership position: Schrodinger Stock ownership: Schrodinger G. Krilov Employment or leadership position: Schrodinger Stock ownership: Schrodinger J. Tan Employment or leadership position: Schrodinger Stock ownership: Schrodinger H. Wright Employment or leadership position: Schrodinger Stock ownership: Schrodinger D. Weiss Employment or leadership position: Schrodinger Stock ownership: Schrodinger W. Yin Employment or leadership position: Schrodinger Stock ownership: Schrodinger K. Akinsanya Employment or leadership position: Schrodinger Stock ownership: Schrodinger

Abstract 4032: Discovery of a potent, selective, and orally available SHP2 inhibitor that can penetrate blood-brain barrier

Abstract Src homology region 2-containing protein tyrosine phosphatase 2 (SHP2/PTPN11) is one of the key signaling proteins in many oncogenic receptor tyrosine kinase (RTK) pathways. It mediates RAS-driven downstream signaling and activates gene expression for tumorigenesis. Importantly, a critical role of SHP2 has been found during the process of temporal adaptation and ERK re-activation after the treatment of RAS/RAF/MEK/ERK inhibitors, which means that SHP2 could be a promising therapeutic target to shut down the oncogenic RAS pathway, especially in combination with RAS/RAF/MEK/ERK inhibitors. Here we present preclinical data of an allosteric small-molecule SHP2 inhibitor that is highly potent, selective, and brain-permeable. In vitro biochemical and cellular assays showed excellent potency against SHP2 protein. In addition, we confirmed exceptional in vivo activity in mouse xenograft models, and demonstrated combination effects with a KRAS G12C inhibitor. Most importantly, the BBB penetration and anti-tumor activity in brain was confirmed both in vitro and in vivo, supporting the great potential in treating brain-metastasized tumors. In summary, we have successfully discovered and developed a potent and blood-brain barrier permeable SHP2 inhibitor that provides a novel therapeutic option for diverse cancers associated in the RAS pathway. Citation Format: Miyeon Kim, Dongsu Kim, Kyeong Jin Yoon, Yeejin Jeon, Dohyun Park, Mijung Lee, Dahye Jeon, Soyeon Jang, Jinhwan Kim, Eunji Kim, Jihoon Park, Victor Hong, Sang Kyun Lim, Sungpil Choi. Discovery of a potent, selective, and orally available SHP2 inhibitor that can penetrate blood-brain barrier. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4032.

Abstract 2791: Biomarker strategy for a phase 1 study of ORIC-944, a potent and selective allosteric PRC2 inhibitor, in patients with metastatic prostate cancer

Abstract Background: Polycomb repressive complex 2 (PRC2) tri-methylates histone H3 at lysine 27 (H3K27me3) leading to transcriptionally silenced genes. ORIC-944 is a potent, highly selective allosteric small molecule inhibitor of PRC2’s embryonic ectoderm development (EED) subunit that is undergoing clinical development as an orally bioavailable monotherapy in patients with metastatic prostate cancer. We previously reported strong tumor growth inhibition with ORIC-944 in enzalutamide-resistant 22Rv1 prostate cancer xenografts. Here, to devise and implement a biomarker strategy aimed at ORIC-944 dose selection in the ongoing phase 1 study (NCT05413421), we undertook preclinical pharmacology studies and assay development. Methods: Naïve and 22Rv1-tumor bearing male mice were treated with vehicle, 10, 30 or 100 mg/kg ORIC-944 PO for up to 37 days. H3K27me3 was evaluated in dorsal skin (epidermal layer) and peripheral monocytes by IHC and an alphaLISA test, respectively. Since dying tumor cells release nucleosomes to circulation, cell-free (cf)-nucleosomal H3K27me3 levels normalized to cf-H3.1 were also assessed in plasma using a proprietary magnetic bead-based sandwich immunoassay. Tumors were profiled by RNA-sequencing and H3K27me3 chromatin immunoprecipitation sequencing. Results: In the epidermal skin layer, H3K27me3 was present in vehicle-treated naïve animals but strongly depleted in ORIC-944-treated mice in a dose-dependent manner. Likewise in monocytes, H3K27me3 was reduced to undetectable levels in 100 mg/kg ORIC-944-treated mice. In plasma, normalized cf-nucleosomal H3K27me3 levels significantly decreased in response to ORIC-944 in a dose- and time-dependent manner, selectively in 22Rv1-tumor bearing mice compared to non-tumor bearing mice. Putative PRC2 target genes were identified in 22Rv1 xenografts by focusing on those genes with H3K27me3 binding in the promoter, whose expression was de-repressed by ORIC-944 in a time-dependent manner and then re-expressed after dose suspension. In the ongoing phase 1 study, H3K27me3 levels are being evaluated in the stratum spinosum of skin and in blood-derived monocytes using a proprietary IHC assay and alphaLISA test, respectively. Modulation in the expression of the putative PRC2 target genes is being assessed in blood-derived peripheral blood mononuclear cells by RNA-sequencing. Conclusion: This comprehensive biomarker strategy enables us to establish target engagement and capture exposure-dependent pharmacodynamics in the ongoing phase 1 study evaluating ORIC-944 as a best-in-class PRC2 inhibitor for the treatment of patients with advanced prostate cancer. Citation Format: Anneleen Daemen, Natalie Yuen, Aleksandr Pankov, Eric A. Ariazi, Subhash D. Katewa, Frank L. Duong, Amber Wang, Shravani Barkund, Shelly Kaushik, Jessica D. Sun, Lori S. Friedman, Melissa R. Junttila. Biomarker strategy for a phase 1 study of ORIC-944, a potent and selective allosteric PRC2 inhibitor, in patients with metastatic prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2791.

Allosteric targeting resolves limitations of earlier LFA-1 directed modalities

Integrins are a family of cell surface receptors well-recognized for their therapeutic potential in a wide range of diseases. However, the development of integrin targeting medications has been impacted by unexpected downstream effects, reflecting originally unforeseen interference with the bidirectional signalling and cross-communication of integrins. We here selected one of the most severely affected target integrins, the integrin lymphocyte function-associated antigen-1 (LFA-1, αLβ2, CD11a/CD18), as a prototypic integrin to systematically assess and overcome these known shortcomings. We employed a two-tiered ligand-based virtual screening approach to identify a novel class of allosteric small molecule inhibitors targeting this integrin’s αI domain. The newly discovered chemical scaffold was derivatized, yielding potent bis-and tris-aryl-bicyclic-succinimides which inhibit LFA-1 in vitro at low nanomolar concentrations. The characterisation of these compounds in comparison to earlier LFA-1 targeting modalities established that the allosteric LFA-1 inhibitors (i) are devoid of partial agonism, (ii) selectively bind LFA-1 versus other integrins, (iii) do not trigger internalization of LFA-1 itself or other integrins and (iv) display oral availability. This profile differentiates the new generation of allosteric LFA-1 inhibitors from previous ligand mimetic-based LFA-1 inhibitors and anti-LFA-1 antibodies, and is projected to support novel immune regulatory regimens selectively targeting the integrin LFA-1. The rigorous computational and experimental assessment schedule described here is designed to be adaptable to the preclinical discovery and development of novel allosterically acting compounds targeting integrins other than LFA-1, providing an exemplary approach for the early characterisation of next generation integrin inhibitors.

A phase 1b study of the allosteric extracellular FGFR2 inhibitor alofanib in patients with pretreated advanced gastric cancer.

Alofanib is a small-molecule allosteric extracellular FGFR2 inhibitor. We report safety and preliminary efficacy from the first-in-human phase 1b study of alofanib in heavily pretreated patients with advanced gastric cancer. The standard dose-escalation design 3+3 aimed to establish the maximum tolerated dose (MTD) or recommended phase 2 dose (RP2D). Alofanib was administered daily intravenously 5 days on, 2 days off. There were five dose levels (50-350 mg/m2). All patients received alofanib until disease progression or unacceptable toxicity. 21 patients were enrolled. Patients were predominantly male (71%), 67% had 2 and more metastatic sites, including liver metastases (43%), 19% had ECOG PS 2, and were heavily pretreated (86% had previous 2 and more treatment lines). During dose escalation, no dose-limiting toxicities were observed, and MTD was not defined. 15 (71.4%) patients had at least one adverse event associated with the treatment (TRAE). Grade 3 or higher TRAEs were observed in 6 patients (28.6%). The most common TRAEs included reactions immediately after administration, diarrhea, thrombocytopenia, arthralgia, and headache. The median progression-free survival and overall survival was 3.63 (95% CI 1.58-5.68) and 7.0 (95% CI 3.82-10.18) months, respectively. The 6- and 12-month overall survival rates were 57.1% and 33.3%. Disease control rate was 68% with one durable partial response. The MTD has not been reached and dose of 350 mg/m2, 5 days on, 2 days off has been declared as RP2D. Alofanib showed acceptable tolerability and preliminary signs of clinical activity in the late-line treatment of metastatic gastric cancer. (ClinicalTrials.gov identifier: NCT04071184).

Small-molecule allosteric inhibitors of GPX4

Encouraged by the dependence of drug-resistant, metastatic cancers on GPX4, we examined biophysical mechanisms of GPX4 inhibition, which revealed an unexpected allosteric site. We found that this site was involved in native regeneration of GPX4 under low glutathione conditions. Covalent binding of inhibitors to this allosteric site caused a conformational change, inhibition of activity, and subsequent cellular GPX4 protein degradation. To verify this site in an unbiased manner, we screened a library of compounds and identified and validated that an additional compound can covalently bind in this allosteric site, inhibiting and degrading GPX4. We determined co-crystal structures of six different inhibitors bound in this site. We have thus identified an allosteric mechanism for small molecules targeting aggressive cancers dependent on GPX4.

Parmodulin, an Allosteric Inhibitor of Protease Activated Receptor 1 (PAR1) Protects Sickle Cell Mice from Thromboinflammation and Endothelial Dysfunction

Protease activated receptor 1 (PAR1) couples the coagulation cascade to endothelial inflammation in many diseases, including sickle cell disease (SCD). PAR1 is a GPCR activated by proteolytic cleavage of an extracellular N-terminus, and demonstrates biased signaling. Activation of PAR1 by thrombin stimulates prothrombotic and proinflammatory signaling, whereas activation by activated protein C (APC) stimulates cytoprotective and anti-inflammatory pathways. The ideal PAR-1 targeted therapy would inhibit detrimental signaling while preserving beneficial signaling. 3K3A-APC is a signaling selective variant of APC that activates cytoprotective PAR1 signaling with minimal anticoagulant effects, demonstrated to have a beneficial role in stroke models. Parmodulin 2 (PM2) is a small molecule allosteric inhibitor of PAR1 that simultaneously blocks detrimental signaling and activate beneficial cytoprotective signaling similar to that induced by APC. We previously demonstrated that endogenous APC-PAR1 signaling contributes to thrombin generation and inflammation in a mouse model of SCD, whereas thrombin-PAR signaling enhances these pathways. We hypothesize that inducing cytoprotective PAR1 signaling with either 3K3A-APC and PM2 will reduce thrombo-inflammation in sickle cell mice at steady state. Townes wild type (HbAA) and sickle (HbSS) mice (male and female, 4 months old) were used in the studies. In the first experiment, HbAA and HbSS (n=4-5 per group) mice were treated daily with either saline (SAL) or 3K3A-APC (0.2mg/kg, BW) for 4 days. Samples were collected 1 hour after final dose. As previously demonstrated, plasma levels of thrombin-anti thrombin (TAT) complexes, the proinflammatory cytokine IL-6, high mobility group box 1 (HMGB1), and, soluble vascular cell adhesion molecule (sVCAM), were increased in HbSS mice compared to HbAA controls. Surprisingly 3K3A-APC treatment did reduce any of these parameters in HbSS mice. APC and its variant 3K3A-APC require binding to the endothelial protein C receptor (EPCR) to activate PAR1; it is known that SCD causes increased EPCR shedding. Indeed, we found that both SAL- and 3K3A-APC treated HbSS mice had elevated sEPCR levels compared to HbAA controls. Together, these data indicate that 3K3A-APC does not affect thrombo-inflammation in HbSS mice at steady state most likely due to by reduced ECPR expression on the endothelium. In the second experiment, HbAA and HbSS (male and female, 4 months old) mice (n=8-11 per group) were treated with vehicle (20% DMSO, 20% PEG800, 60% PBS) or PM2 (10 mg/kg, BW) every other day for 1 week. The last dose was administered on the 8th day 1 hour prior to collecting plasma samples. As expected, HbSS mice had elevated plasma TAT levels compared to HbAA mice (11.04 ± 1.4 vs 45.19 ± 6.3, p<0.05) which was significantly attenuated by PM2 treatment in SS mice (16.17 ± 2, p<0.05). Similar results were observed for IL-6 (AA: 2.06 ± 0.7 vs SS: 29.12 ± 9.6 vs SS+PM2: 5.83 ± 2.9; p<0.05); sVCAM (AA: 330.6 ± 31.5 vs SS: 571.4 ± 50.4 vs SS+PM2: 403.3 ± 41.22; p<0.05) and HMGB1 (AA: 11.46 ± 1.1 vs SS: 25.37 ± 1.5 vs SS+PM2: 19.12 ± 1.7; p<0.05). PM2 had no effect on markers of anemia in HbSS mice including RBC, hematocrit, and reticulocyte counts. In the present study ,3K3A-APC did not attenuate thrombin generation and cytokine production in HbSS mice; this might be due to the shedding of EPCR which is important co-receptor for the protective APC-PAR1 signaling. In contrast, PM2-mediated allosteric inhibition of thrombin/PAR1 and simultaneous induction of APC-like cytoprotective signaling attenuated levels of TAT, IL-6, HMGB1, and sVCAM in HbSS mice. Future studies towards evaluating the effect of PM2 on downstream targets of PAR1 signaling in sickle mice will provide more insight into the mechanism of this protective effect.

BDK inhibition acts as a catabolic switch to mimic fasting and improve metabolism in mice

ObjectiveBranched chain amino acid (BCAA) catabolic defects are implicated to be causal determinates of multiple diseases. This work aimed to better understand how enhancing BCAA catabolism affected metabolic homeostasis as well as the mechanisms underlying these improvements. MethodsThe rate limiting step of BCAA catabolism is the irreversible decarboxylation by the branched chain ketoacid dehydrogenase (BCKDH) enzyme complex, which is post-translationally controlled through phosphorylation by BCKDH kinase (BDK). This study utilized BT2, a small molecule allosteric inhibitor of BDK, in multiple mouse models of metabolic dysfunction and NAFLD including the high fat diet (HFD) model with acute and chronic treatment paradigms, the choline deficient and methionine minimal high fat diet (CDAHFD) model, and the low-density lipoprotein receptor null mouse model (Ldlr−/−). shRNA was additionally used to knock down BDK in liver to elucidate liver-specific effects of BDK inhibition in HFD-fed mice. ResultsA rapid improvement in insulin sensitivity was observed in HFD-fed and lean mice after BT2 treatment. Resistance to steatosis was assessed in HFD-fed mice, CDAHFD-fed mice, and Ldlr−/− mice. In all cases, BT2 treatment reduced steatosis and/or inflammation. Fasting and refeeding demonstrated a lack of response to feeding-induced changes in plasma metabolites including insulin and beta-hydroxybutyrate and hepatic gene changes in BT2-treated mice. Mechanistically, BT2 treatment acutely altered the expression of genes involved in fatty acid oxidation and lipogenesis in liver, and upstream regulator analysis suggested that BT2 treatment activated PPARα. However, BT2 did not directly activate PPARα in vitro. Conversely, shRNA-AAV-mediated knockdown of BDK specifically in liver in vivo did not demonstrate any effects on glycemia, steatosis, or PPARα-mediated gene expression in mice. ConclusionsThese data suggest that BT2 treatment acutely improves metabolism and liver steatosis in multiple mouse models. While many molecular changes occur in liver in BT2-treated mice, these changes were not observed in mice with AAV-mediated shRNA knockdown of BDK. All together, these data suggest that systemic BDK inhibition is required to improve metabolism and steatosis by prolonging a fasting signature in a paracrine manner. Therefore, BCAA may act as a “fed signal” to promote nutrient storage and reduced systemic BCAA levels as shown in this study via BDK inhibition may act as a “fasting signal” to prolong the catabolic state.

Discovery of Small-Molecule Allosteric Inhibitors of PfATC as Antimalarials.

The discovery and development of new drugs against malaria remain urgent. Aspartate transcarbamoylase (ATC) has been suggested to be a promising target for antimalarial drug development. Here, we describe a series of small-molecule inhibitors of P. falciparum ATC with low nanomolar binding affinities that selectively bind to a previously unreported allosteric pocket, thereby inhibiting ATC activation. We demonstrate that the buried allosteric pocket is located close to the traditional ATC active site and that reported compounds maintain the active site of PfATC in its low substrate affinity/low activity conformation. These compounds inhibit parasite growth in blood stage cultures at single digit micromolar concentrations, whereas limited effects were seen against human normal lymphocytes. To our knowledge, this series represent the first PfATC-specific allosteric inhibitors.

99 (PB089) - Discovery of an allosteric small molecule inhibitor that can potently target SHP2 in vitro and in vivo

Background: SHP2 (Src homology region 2-containing protein tyrosine phosphatase 2) is the only known tumor-promoting phosphatase, that relays the signals from the diverse receptor tyrosine kinases to the RAS/MAPK pathway. Abnormal SHP2 activation and activating mutations are frequently observed in various cancers and accordingly genetic or pharmacological inhibition of SHP2 has been reported to suppress RTK/KRAS-mediated cancer progression. Moreover, SHP2 serves as a key component of the ERK re-activation in the resistance mechanism to KRAS pathway inhibitors. Therefore, SHP2 inhibitor has a great therapeutic potential in treating RTK/ RAS-mediated cancer as monotherapy and in combination treatment. Material and methods: Biochemical phosphatase assays were performed by detecting DiFMU which is converted by SHP2(WT or mutants) with various concentrations of compounds. For the cellular pERK analysis, compounds were treated into H358 cells followed by pERK measurement using the AlphaLISA system (PerkinElmer). Cell viability was measured by CellTiter-Glo (Promega) after 3-day treatment with compounds. For the in vivo tumor model, cancer cells were inoculated into the flank region of BALB/ c nude mice. Once the tumor size reached 100 mm3, compounds were administrated (P.O.,Q.D). Tumor size was measured twice a week. Results: Cpd1, an orally available and highly potent SHP2 inhibitor, profoundly suppressed SHP2 activity in vitro and KRAS-mutated tumor growth in mouse xenograft models, outcompeting the positive control TNO-155. In detail, Cpd1 potently inhibited SHP2(WT) and also the highly-active oncogenic SHP2 mutants. It impeded the proliferation of cancer cells encoding KRAS and/or SHP2 mutations. With an excellent PK profile, Cpd1 induced in vivo tumor regression in KRAS mutation-driven models. Conclusions: Cpd1 is a potent and selective SHP2 allosteric inhibitor, with a great anti-tumor effect in vitro and in vivo. It may provide a novel therapeutic option for diverse cancers, including KRAS-driven tumors, as a monotherapy or in combination therapies. No conflict of interest.

PH domain-mediated autoinhibition and oncogenic activation of Akt.

Akt is a Ser/Thr protein kinase that plays a central role in metabolism and cancer. Regulation of Akt's activity involves an autoinhibitory intramolecular interaction between its pleckstrin homology (PH) domain and its kinase domain that can be relieved by C-tail phosphorylation. PH domain mutant E17K Akt is a well-established oncogene. Previously, we reported that the conformation of autoinhibited Akt may be shifted by small molecule allosteric inhibitors limiting the mechanistic insights from existing X-ray structures that have relied on such compounds (Chu et al., 2020). Here, we discover unexpectedly that a single mutation R86A Akt exhibits intensified autoinhibitory features with enhanced PH domain-kinase domain affinity. Structural and biochemical analysis uncovers the importance of a key interaction network involving Arg86, Glu17, and Tyr18 that controls Akt conformation and activity. Our studies also shed light on the molecular basis for E17K Akt activation as an oncogenic driver.

Pharmacologic inhibition of discoidin domain Receptor 2 (DDR2) sensitizes homologous recombination proficient ovarian cancer models to treatment with olaparib (245)

Pharmacologic inhibition of discoidin domain Receptor 2 (DDR2) sensitizes homologous recombination proficient ovarian cancer models to treatment with olaparib (245)

Hypertrophic cardiomyopathy: A modern view on the diagnosis and management of patients (review)

Hypertrophic cardiomyopathy (HCM) is a genetically determined disease with a high prevalence and manifestation at the age of 30–40 years. Currently available most effective treatments are extended myectomy and Morrow septal myectomy. However, the frequent occurrence of postoperative complications and restrictions to the use of these methods in certain groups of patients provides rationale for the improvement of the existing treatment methods and search for new pharmacological approaches. One of the most promising areas of conservative therapy is the study of a specific small-molecule allosteric inhibitor of myosinadenosine triphosphatase (mavacamten). Clinical studies of the efficacy and safety of this drug continue to this day, and if they are successfully completed, the drug may be included in the pharmacotherapy protocol for HCM.