Class Of Small-molecule Inhibitors Research Articles

MCL1 inhibition targets Myeloid Derived Suppressors Cells, promotes antitumor immunity and enhances the efficacy of immune checkpoint blockade

Immune checkpoint inhibitors (ICIs) are now the first-line treatment for patients with advanced melanoma. Despite promising clinical results, many patients fail to respond to these therapies. BH3 mimetics, a novel class of small molecule inhibitors that bind and inhibit anti-apoptotic members of the BCL2 family proteins such as BCL2 or MCL1, have been very successful in treating hematologic malignancies. However, there are limited studies on the immunomodulatory role of the BH3 mimetics. Several factors contribute to ICI resistance including myeloid-derived suppressor cells (MDSCs) that exert immunosuppressive effects through direct and indirect inhibition of antitumor immunity. Thus, targeting MDSCs to enhance antitumor immunity has the potential to enhance the efficacy of ICIs. In this study, we show that the MCL1 inhibitor S64315 reduces melanoma tumor growth in an immune cell-dependent manner in mice. Specifically, S64315 enhances antitumor immunity by reducing MDSC frequency and by promoting the activity of CD8+T cells. Additionally, human MDSCs are 10 times more sensitive to S64315 than cutaneous melanoma lines. Further, we found that a higher expression of MCL1 is associated with poor survival for patients treated with anti-PD-1. Finally, combining S64315 and anti-PD-1 significantly slowed tumor growth compared to either agent alone. Together, this proof-of-concept study demonstrates the potential of combining an MCL1 inhibitor with anti-PD-1 in the treatment of melanoma. It justifies the further development of next generation MCL1 inhibitors to improve efficacy of ICIs in treating malignant melanoma.

Structure-based design and optimization of a new class of small molecule inhibitors targeting the P-stalk binding pocket of ricin

Ricin, a category-B agent for bioterrorism, and Shiga toxins (Stxs), which cause food poisoning bind to the ribosomal P-stalk to depurinate the sarcin/ricin loop. No effective therapy exists for ricin or Stx intoxication. Ribosome binding sites of the toxins have not been targeted by small molecules. We previously identified CC10501, which inhibits toxin activity by binding the P-stalk pocket of ricin toxin A subunit (RTA) remote from the catalytic site. Here, we developed a fluorescence polarization assay and identified a new class of compounds, which bind P-stalk pocket of RTA with higher affinity and inhibit catalytic activity with submicromolar potency. A lead compound, RU-NT-206, bound P-stalk pocket of RTA with similar affinity as a five-fold larger P-stalk peptide and protected cells against ricin and Stx2 holotoxins for the first time. These results validate the P-stalk binding site of RTA as a critical target for allosteric inhibition of the active site.

Dipeptidyl peptidase 4 inhibitor reduces tumor-associated macrophages and enhances anti-PD-L1-mediated tumor suppression in non-small cell lung cancer

PurposeThe efficacy of immune checkpoint inhibitors such as programmed cell death ligand 1 (PD-L1) antibodies in non-small cell lung cancer (NSCLC) is limited, and combined use with other therapies is recommended. Dipeptidyl peptidase 4 (DPP4) inhibitors, a class of small molecule inhibitors, are highly effective for treating type 2 diabetes. Emerging evidence implicates DPP4 inhibitors as immunomodulators that modify aspects of innate and adaptive immunity. We evaluated the combination of a DPP4 inhibitor (anagliptin) and PD-L1 blockade in an NSCLC mouse model.MethodsThe effect of the combination of anti-PD-L1 and anagliptin was evaluated in subcutaneous mouse models of NSCLC. Tumor-infiltrating immune cells were analyzed by flow cytometry. Bone marrow-derived monocytes of C57BL/6 mice were isolated in vitro to examine the underlying mechanism of anagliptin on the differentiation and polarization of macrophage.ResultsAnagliptin dramatically improved the efficacy of PD-L1 antibody monotherapy by inhibiting macrophage formation and M2 polarization in the tumor microenvironment. Mechanistically, anagliptin suppressed the production of reactive oxygen species in bone marrow monocytes by inhibiting NOX1 and NOX2 expression induced by macrophage colony-stimulating factor, reduced late ERK signaling pathway activation, and inhibited monocyte-macrophage differentiation. However, the inhibitory effect was reactivated by lipopolysaccharide and interferon-gamma interacting with corresponding receptors during M1 macrophage polarization, but not M2.ConclusionsAnagliptin can enhance PD-L1 blockade efficacy in NSCLC by inhibiting macrophage differentiation and M2 macrophage polarization, and combination therapy may be a promising strategy for treating PD-L1 blockade therapy-resistant patients with NSCLC.

TMPRSS2 and SARS-CoV-2 SPIKE interaction assay for uHTS.

SARS-CoV-2, the coronavirus that causes the disease COVID-19, has claimed millions of lives over the past 2 years. This demands rapid development of effective therapeutic agents that target various phases of the viral replication cycle. The interaction between host transmembrane serine protease 2 (TMPRSS2) and viral SPIKE protein is an important initial step in SARS-CoV-2 infection, offering an opportunity for therapeutic development of viral entry inhibitors. Here, we report the development of a time-resolved fluorescence/Förster resonance energy transfer (TR-FRET) assay for monitoring the TMPRSS2-SPIKE interaction in lysate from cells co-expressing these proteins. The assay was configured in a 384-well-plate format for high-throughput screening with robust assay performance. To enable large-scale compound screening, we further miniaturized the assay into 1536-well ultrahigh-throughput screening (uHTS) format. A pilot screen demonstrated the utilization of the assay for uHTS. Our optimized TR-FRET uHTS assay provides an enabling platform for expanded screening campaigns to discover new classes of small-molecule inhibitors that target the SPIKE and TMPRSS2 protein-protein interaction.

Janus Kinase Inhibitors: A Promising Therapeutic Option for Allergic Contact Dermatitis

Janus kinase (JAK) inhibitors represent a promising class of small molecule inhibitors that treat a range of inflammatory skin diseases, such as atopic dermatitis (AD), psoriasis, and alopecia areata. Although the evidence for their use in allergic contact dermatitis (ACD) remains limited, early results from animal studies and case reports are promising. Herein, we provide an overview of JAK inhibitors and the evidence for their use in ACD.

Identification of natural inhibitor from Aframomum melegueta targeting survivin and mammalian rapamycin signaling pathway in Kidney Cancer

Overactivation of survivin and mammalian rapamycin signaling pathway plays an important role in renal carcinogenesis. Recent studies have shown that extract from Aframomum species produces interesting cytotoxic effects against drug resistance cancer. However, the effect of Aframomum melegueta on renal survivin and mammalian rapamycin signaling pathway have not been studied. In this study, we have described a thorough computational study on the effect of natural compounds from Aframomum melegueta on survivin and mammalian rapamycin target at molecular level. Our results revealed a new class of small molecule inhibitors, namely {1,3}benzodaoxolo{5,6-c} dihydrogingerenone, 5-hydroxy-7-methoxyflavone, apigenin, 3,4-dimethoxyphenylacetone and arctigenin, which effectively inhibit survivin and mammalian rapamycin signaling activity. Furthermore, the ADMET (Adsorption, distribution, metabolism, excretion and toxicological) properties prediction also shows that these compounds had good pharmacological properties with little or no toxicity effect. Collectively, our finding suggests that the natural compounds from Aframomum melegueta reported in this study could serve as a potent therapeutic inhibitor of survivin and mammalian rapamycin in renal cancer cells. Thus, we propose further investigation of these compound to validate our findings.

Functional Anti-Apoptotic Protein Dependence and Its Relation to Genomic Prognostic Markers and Response to Treatment in Chronic Lymphocytic Leukemia

Introduction Due to its heterogenous biology, chronic lymphocytic leukemia (CLL) is a well-suited disease for identifying new relationships between genomic and functional biomarkers for drug response. Here, we combined a multi-omics approach and ex vivo drug-treatment data with functional characterization of apoptotic sensitivity to correlate genetic features and drug sensitivity with specific anti-apoptotic signaling patterns measured by BH3 profiling in primary CLL samples. Methods BH3 profiling is a functional assay to measure the proximity of cells to undergo apoptosis via overall priming as well as to identify specific anti-apoptotic proteins the cells are dependent on for survival. BH3 peptides and mimetic such as BIM and PUMA were used to identify overall priming or proximity to apoptosis, BAD and ABT199 were used to identify BCL-2 dependence, and FS1, MS1 and HRK were used to identify BFL-1, MCL-1 and BCL-xL dependences respectively (Ryan et al, Biol Chem, 2016). Results We analyzed primary CLL samples from 73 patients and using BH3 profiling identified the specific associations between anti-apoptotic protein dependence and genetic features (such as IGHV status, trisomy-12, del(17p) and TP53 mutation. Our univariate models demonstrated that BCL-2 dependence, as measured by ABT199 drug (p=0.001) and BAD (p=0.004) BH3 peptide, are highly correlated with mutated IGHV status, a favorable prognostic marker in CLL (Figure 1A). Using ex vivo treatment with multiple classes of small molecule inhibitors, we further identified several specific drug response or sensitivity patterns correlating with anti-apoptotic protein dependence as well as CLL prognostic factors (Figure 1B). In particular, our multivariate models (LASSO regularized) demonstrated that viability following ex vivo treatment with the MDM-2 inhibitor Nutlin-3 was positively correlated with TP53 mutation/deletion, but inversely correlated with BFL-1 (FS1 peptide) and BCL-xL (HRK peptide) dependence as well as trisomy-12 (Figure 1B), though the inverse correlation between Nutlin-3 and BFL-1 or BCL-xL dependence is relatively weak. CLL cell viability following ex vivo treatment with the dual BCL-2/BCL-xL inhibitor navitoclax was negatively correlated with BCL-2 (BAD peptide) and BCL-xL (HRK peptide) dependence, though primarily BCL-2 dependence, suggesting that sensitivity to navitoclax is highly dependent on BCL-2 (Figure 1B). At the transcriptomic level, CLL cell dependence on BCL-2 positively correlated with NOXA expression, NFκB signaling, and reduced intracellular oxidative level, various pathways that could potentially maintain BCL-2 dependence. Finally, we utilized BH3 profiling to retrospectively predict in vivo drug response, with high BCL-2 dependence (p=0.016) correlating with greater absolute lymphocyte count decrease. Conclusion Functional characterization of apoptotic signaling in CLL demonstrated that patients with mutated IGHV are more dependent on BCL-2 for survival, and that BH3 profiling could retrospectively predict in vivo sensitivity to BCL-2 inhibition. This functional precision medicine approach may help to stratify patients with CLL beyond genetic markers and should be further explored for correlation with clinical outcomes in prospective clinical trials. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

BH3 mimetics in combination with nilotinib or ponatinib represent a promising therapeutic strategy in blast phase chronic myeloid leukemia

Dysregulation of the BCL-2 family is implicated in protecting chronic myeloid leukemia (CML) cells from intracellular damage and BCR::ABL1-inhibition with tyrosine kinase inhibitors (TKIs) and may be a viable therapeutic target in blast phase (BP-)CML, for which treatment options are limited. BH3 mimetics, a class of small molecule inhibitors with high-specificity against the prosurvival members of the BCL-2 family, have displayed clinical promise in the treatment of chronic lymphocytic and acute myeloid leukemia as single agents and in combination with standard-of-care therapies. Here we present the first comparison of inhibition of BCL-2 prosurvival proteins BCL-2, BCL-xL and MCL-1 in combination with a second or third generation TKI, crucially with comparisons drawn between myeloid and lymphoid BP-CML samples. Co-treatment of four BP-CML cell lines with the TKIs nilotinib or ponatinib and either BCL-2 (venetoclax), MCL-1 (S63845) or BCL-xL (A-1331852) inhibitors resulted in a synergistic reduction in cell viability and increase in phosphatidylserine (PS) presentation. Nilotinib with BH3 mimetic combinations in myeloid BP-CML patient samples triggered increased induction of apoptosis over nilotinib alone, and a reduction in colony-forming capacity and CD34+ fraction, while this was not the case for lymphoid BP-CML samples tested. While some heterogeneity in apoptotic response was observed between cell lines and BP-CML patient samples, the combination of BCL-xL and BCR::ABL1 inhibition was consistently effective in inducing substantial apoptosis. Further, while BH3 mimetics showed little efficacy as single agents, dual-inhibition of BCL-2 prosurvival proteins dramatically induced apoptosis in all cell lines tested and in myeloid BP-CML patient samples compared to healthy donor samples. Gene expression and protein level analysis suggests a protective upregulation of alternative BCL-2 prosurvival proteins in response to BH3 mimetic single-treatment in BP-CML. Our results suggest that BH3 mimetics represent an interesting avenue for further exploration in myeloid BP-CML, for which alternative treatment options are desperately sought.

Effect of Mcl-1 Inhibitor, AMG-176, on T Cell Quality and Function

Introduction AMG-176 belongs to a class of small molecule inhibitors known as BH3 mimetics, which mimic the role of BH3-only proteins and inhibit pro-survival Bcl-2 family proteins. AMG-176 specifically inhibits the pro-survival Mcl-1 protein. Overexpression of anti-apoptotic protein Mcl-1 has been extensively reported in hematological malignancies including mantle cell lymphomas (MCL), diffuse large B cell lymphomas (DLBCL), and multiple myeloma (MM). Chromosomal aberration of Mcl1 was identified as one of the most important survival genes for MM, follicular lymphoma (FL), MCL, and DLBCL, and is associated with shorter patient survival. These hematological malignancies are targetable by FDA-approved chimeric antigen receptor (CAR) T cell therapies with varying efficacies as single agent regimens. The role of Mcl-1 in T cell biology could be further elucidated to improve therapeutic outcomes of malignancies characterized by Mcl-1 overexpression. Here, our team explores the effect of Mcl-1 inhibitor, AMG-176, on T cell quality and function. Methods Our lab obtained healthy human donor blood from consenting adults, then isolated pan T cells using microbead negative selection (Miltenyi Biotec). Following isolation, T cells were cryopreserved in cryopreservative medium consisting of 90% fetal bovine serum (Corning) and 10% dimethyl sulfoxide (Sigma-Aldrich). Upon thaw, T cells were cultured in complete RPMI supplemented with [10ng/mL] h-IL-7 (Peprotech) and [100IU/mL] h-IL-2, and activated for 24 hours using αCD3/αCD28 coated beads (Thermofisher). At 24 hours post-activation, cells were exposed to various concentrations of AMG-176 (MedChem Express). Following 72-hour exposure, T cells were harvested and assessed for ATP production, viability, proliferation, immunophenotype, and effector function. To determine ATP production, we used a commercially available luminescence-based assay (Promega) and obtained our readout using a BioTek Synergy HTX plate reader, T cell immunophenotype was identified following 72-hour exposure to AMG-176 at 0nM, 10nM, 100nM, 500nM, and 1uM concentrations. Samples from each cohort were stained with Live/dead yellow viability dye, CD3, CD4, CD8, CD69, CD25, and CD127, then acquired using a BD LSRII flow cytometer. Flow cytometry data was analyzed using FCS Express analytical software (De Novo Software). Intracellular cytokine staining was performed using markers for Granzyme B, Perforin, and IFN-γ. Results T cells exposed in vitro to 10nM and 100nM concentrations of AMG-176 exhibited greater ATP production (Fig 1.), viability, and proliferation (Fig. 2) than the unexposed control group. T cells exposed to 500nM or 1uM concentrations of AMG-176 exhibited unchanged or decreased ATP production, viability, and proliferation relative to the unexposed control group. T cells exposed to AMG-176 significantly increased CD8+ T cell frequency at 10nM and 100nM concentrations (Fig. 3), without affecting viability of CD3+ Pan T cells. Our flow data also indicates dose-dependent increase in expression of early activation marker CD69+. Conclusion Exposure of human T cells to Mcl-1 inhibitor, AMG-176, provides an increase in ATP production, viability, proliferation, and activation. These results shed light on the role of Mcl-1 in T cell biology. Furthermore, increases in human T cell viability, proliferation, and function as facilitated by AMG-176 could offer insight into the advancement of effector cell-based therapies, such as chimeric antigen receptor (CAR) therapy. We may employ AMG-176 alongside CAR T therapy in order to achieve more effective clinical outcomes. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A comprehensive review of SHP2 and its role in cancer

Src homology 2-containing protein tyrosine phosphatase 2 (SHP2) is a non-receptor protein tyrosine phosphatase ubiquitously expressed mainly in the cytoplasm of several tissues. SHP2 modulates diverse cell signaling events that control metabolism, cell growth, differentiation, cell migration, transcription and oncogenic transformation. It interacts with diverse molecules in the cell, and regulates key signaling events including RAS/ERK, PI3K/AKT, JAK/STAT and PD-1 pathways downstream of several receptor tyrosine kinases (RTKs) upon stimulation by growth factors and cytokines. SHP2 acts as both a phosphatase and a scaffold, and plays prominently oncogenic functions but can be tumor suppressor in a context-dependent manner. It typically acts as a positive regulator of RTKs signaling with some inhibitory functions reported as well. SHP2 expression and activity is regulated by such factors as allosteric autoinhibition, microRNAs, ubiquitination and SUMOylation. Dysregulation of SHP2 expression or activity causes many developmental diseases, and hematological and solid tumors. Moreover, upregulated SHP2 expression or activity also decreases sensitivity of cancer cells to anticancer drugs. SHP2 is now considered as a compelling anticancer drug target and several classes of SHP2 inhibitors with different mode of action are developed with some already in clinical trial phases. Moreover, novel SHP2 substrates and functions are rapidly growing both in cell and cancer. In view of this, we comprehensively and thoroughly reviewed literatures about SHP2 regulatory mechanisms, substrates and binding partners, biological functions, roles in human cancers, and different classes of small molecule inhibitors target this oncoprotein in cancer.

EZH2 Inhibitors Suppress Colorectal Cancer by Regulating Macrophage Polarization in the Tumor Microenvironment

EZH2 inhibitors (EZH2i), a class of small-molecule inhibitors that target EZH2 to exert anti-tumor functions, have just been approved by the US Food and Drug Administration (FDA) in treatment of adults and adolescents with locally advanced or metastatic epithelioid sarcoma. The application of EZH2i in several solid tumors is still in different stages of clinical trials and needs to be further validated. As a key epigenetic regulator, besides its role in controlling the proliferation of tumor cells, EZH2 has been implicated in the regulation of various immune cells including macrophages. But there are still controversial research results at present. Colorectal cancer (CRC) is a common malignant tumor that highly expresses EZH2, which has the third highest incidence and is the second leading cause of cancer-related death worldwide. Studies have shown that the numbers of M2-type tumor-associated macrophages (TAMs) are highly associated with the progression and metastasis of CRC. In the current study, we aim to investigate how EZH2 modulates the polarization of macrophages in the tumor microenvironment (TME) of CRC, and compare the role of two different EZH2 inhibitors, EPZ6438 and GSK126. We applied a 3D culture method to demonstrate that EZH2i did indeed suppress the proliferation of CRC cells in vitro. In vivo, we found that the percentage of CD206+ macrophages of the TME was decreased under the treatment of EPZ6438, but it increased upon GSK126 treatment. Besides, in the co-culture system of macrophages and CRC cells, EPZ6438 led to significant elevation of M1 markers and reduction of M2 markers. Furthermore, mechanistic studies validated by ChIP-qPCR demonstrated that EZH2i inhibit EZH2-mediated H3K27me3 levels on the promoters of STAT3, an essential transcription factor for M1 macrophage polarization. Therefore, our data suggested that EZH2i not only suppress CRC cell proliferation directly, but also regulate macrophage by skewing M2 into effector M1 macrophage to exert a tumor suppressive effect. Moreover, our study provided new insight for better understanding of the role of two kinds of EZH2i: EPZ6438 and GSK126, which may pave the way in treating CRC by targeting cancer cells and immune cells via this epigenetic approach in the future.

HBI-8000, HUYABIO Lead Clinical Program, Is a Selective Histone Deacetylase Inhibitor With Therapeutic Benefits in Leukemia and in Solid Tumors.

HBI-8000 is a small molecule inhibitor of class I HDACs and has been approved for the treatment of PTCL, ATL and, in combination with exemestane, in a subpopulation of breast cancer. Given the roles of HDACs in normal and cancerous cells, there are currently multiple clinical trials, by HUYABIO International, to test the efficacy of HBI-8000 in monotherapy or in combination settings in leukemias and in solid tumors. The current review is focused on the applications of HDACi HBI-8000 in cancer therapy and its potential in combination with DDR agents.

Novel 1,2,4-oxadiazole/pyrrolidine hybrids as DNA gyrase and topoisomerase IV inhibitors with potential antibacterial activity

DNA gyrase is a promising target for antibacterial agents. Several classes of small-molecule inhibitors have been discovered in recent decades, but none of these have reached the market. We have designed a small library of 1,2,4-oxadiazole/pyrrolidine hybrids with mid nanomolar inhibitory and potent antibacterial activities against DNA gyrase and topoisomerase IV. Compounds 9 , 15 , 16 , 19 , and 21 inhibited Escherichia coli DNA gyrase to a similar extent as the reference compound, novobiocin, with inhibitory values ranging from 120 nM to 270 nM. Compound 16 was one of the most potent compounds in the series, with an IC 50 value of 120 nM against E. coli gyrase, which is lower than the IC 50 value of novobiocin (170 nM). Compound 16 had the highest inhibitory activity, with minimum inhibitory concentrations (MIC) of 24 and 62 ng/mL against Staphylococcus aureus and E. coli , respectively, which compared favorably with ciprofloxacin (30 and 60 ng/mL, respectively). Compounds 9 , 15 , 19 , and 21 were similar to novobiocin in terms of their activity against E. coli and S. aureus topoisomerase IV, while compound 16 was more potent than novobiocin.

Synthesis of copper (II) porphyrin complexes and their interaction with c-myc G-quadruplex DNA

ObjectiveTo synthesize copper (II) porphyrin complexes and study their interactions with c-myc G-quadruplex DNA. MethodsThe 5-p-hydroxyphenyl-10, 15, 20-tris (p-methoxyphenyl) copper (II) porphyrin complex [p-HTMOPPCu (II)] was synthesized by the conventional heating method. Ultraviolet (UV) titration, fluorescence titration, fluorescence resonance energy transfer (FRET) melting point and competitive assays were used to study the interactions between p-HTMOPPCu (II) and c-myc G-quadruplex DNA. ResultsThe UV absorption spectrum and fluorescence spectroscopy results indicated that the complex of p-HTMOPPCu (II) bound better with c-myc G-quadruplex DNA; the FRET melting point assay, and competitive melting point assay demonstrated that p-HTMOPPCu (II) could selectively bind and stabilize c-myc G-quadruplex DNA. Conclusionp-HTMOPPCu (II) can bind and stabilize c-myc G-quadruplex DNA and will potentially be developed into a class of small molecule inhibitors targeting c-myc G-quadruplex DNA for clinical applications in the treatment of tumors.

Rational Design and Development of HDAC Inhibitors for Breast Cancer Treatment

Breast cancer is the most prevalent cancer amongst females across the globe, and with over 2 million new cases reported in 2018, it poses a huge economic burden to the already dwindling public health. A dearth of therapies in the pipeline to treat triple-negative breast cancers and acquisition of resistance against the existing line of treatments urge the need to strategize novel therapeutics in order to add new drugs to the pipeline. HDAC inhibitors (HDACi) is one such class of small molecule inhibitors that target histone deacetylases to bring about chromosomal remodelling and normalize dysregulated gene expression that marks breast cancer progression. While four HDACi have been approved by the FDA for the treatment of different cancer types, no HDACi is specifically earmarked for clinical management of breast cancer. Owing to the differential HDAC expression pertaining to different types of breast cancers, isoform-selective HDAC inhibitors need to be discovered. This review attempts to set the stage for the rational structure-based discovery of isoform-selective HDACi by providing structural insights into different HDACs and their catalytic folds based on their classes and individual landscape. The development of inhibitors in accordance with the differential expression of HDAC isoforms exhibited in breast cancer cells is a promising strategy to rationally design selective and effective inhibitors, adopting a 'personalized-medicine' approach.

Case Presentation – Frontline Treatment of Older Patient with CLL: Options and Consideration

AbstractThe advent of novel targeted therapies, specifically Bruton’s tyrosine kinase (BTK) inhibitors and the B-cell lymphoma 2 (BCL2) inhibitor, venetoclax, has revolutionized the treatment approach in the frontline setting for patients with chronic lymphocytic leukemia (CLL). By targeting BTK, a critical kinase in proximal B-cell receptor (BCR) signaling, this class of small molecule inhibitors impairs BCR-signaling and activation of NF-κB, and inhibits cell proliferation and migration.1,2 BCL2 is an antiapoptotic protein that is overexpressed in various B-cell cancers, including CLL.3 Venetoclax disrupts antiapoptotic signaling through BCL2, thereby inducing programmed cell death of CLL cells.4 Both of these options have significantly improved the efficacy of treatment in older patients in which efficacy of more traditional chemoimmunotherapy regimens was often hampered by increased toxicity limiting therapy. When embarking on initial therapy for patients with CLL and deciding between various options, it has become important to consider age, comorbidities, and functional condition as well as their disease specific features such as the results of their FISH and IGHV mutational status. Armed with this information, a provider can then review potential options of therapy with each patient focusing on both their disease specific features and comorbidities while taking into consideration their desire for continuous treatment versus time-limited therapy.

A Novel Inhibitor Targeting NLRP3 Inflammasome Reduces Neuropathology and Improves Cognitive Function in Alzheimer's Disease Transgenic Mice.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder, and the most common type of dementia. A growing body of evidence has implicated neuroinflammation as an essential player in the etiology of AD. Inflammasomes are intracellular multiprotein complexes and essential components of innate immunity in response to pathogen- and danger-associated molecular patterns. Among the known inflammasomes, the NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome plays a critical role in the pathogenesis of AD. We recently developed a novel class of small molecule inhibitors that selectively target the NLRP3 inflammasome. One of the lead compounds, JC124, has shown therapeutic efficacy in a transgenic animal model of AD. In this study we tested the preventative efficacy of JC124 in another strain of transgenic AD mice. In this study, 5-month-old female APP/PS1 and matched wild type mice were treated orally with JC124 for 3 months. After completion of treatment, cognitive functions and AD pathologies, as well as protein expression levels of synaptic proteins, were assessed. We found that inhibition of NLRP3 inflammasome with JC124 significantly decreased multiple AD pathologies in APP/PS1 mice, including amyloid-β (Aβ) load, neuroinflammation, and neuronal cell cycle re-entry, accompanied by preserved synaptic plasticity with higher expression of pre- and post-synaptic proteins, increased hippocampal neurogenesis, and improved cognitive functions. Our study demonstrates the importance of the NLRP3 inflammasome in AD pathological development, and pharmacological inhibition of NLRP3 inflammasome with small molecule inhibitors represents a potential therapy for AD.

Abstract LB125: Pharmacokinetics of paxalisib in phase 2 clinical study in glioblastoma (GBM) with unmethylated O6-methylguanine-methyltransferase (MGMT) promotor status

Abstract Introduction: Paxalisib is a potent, oral, selective, brain-penetrant, small molecule inhibitor of class I phosphoinositide 3-kinase and mammalian target of rapamycin. Paxalisib is taken daily on a continuous basis in 28-day cycles. A phase I study (NCT01547S46) in recurrent high-grade gliomas identified an MTD of 45mg daily and is complete. This phase II study (NCT03522298) in patients with newly diagnosed GBM and an unmethylated MGMT promotor identified an MTD of 60mg daily and is now fully recruited and in follow-up. The phase I study showed paxalisib was rapidly absorbed (Tmax approx 2 hours) and displayed an approximately linear and dose proportional increase in Cmax and AUC0-24, with a half-life of approximately 18.7 hours, which is supportive of once-daily dosing. This study explores pharmacokinetics in a slightly different patient population, at an increased dose, and explores the effect of food. Methods: For all patients, on Day 1 of Cycle 1, plasma samples were collected at least 15 minutes prior to the visit dose, and then at 30 minutes, 1, 2, 3, 4, 6, 8, 24 hours post dose. In the escalation portion of the study (Stage 1), patients fasted for 10 hours pre-dose on PK days. In the expansion portion of this study (Stage 2), subjects were randomized to be fasted for 10 hours pre-dose or consume a high-fat, high-calorie meal approximately 30 minutes prior to dosing, on PK days. All samples were analyzed by a central laboratory using a validated LC/MS-MS method. Analysis was non-compartmental. Results: A total of 30 patients were dosed and PK analysis performed, 24 at the 60 mg dose (14 fasted/10 fed) and 6 at the 75mg dose (all fasted). Paxalisib was rapidly and steadily absorbed at the 60 mg and 75 mg dose levels in the fasted state, with median Tmax values of 3.50 (2-8) and 2.50 (2-4) hours post-dose respectively. The geometric mean (range) of t1/2 values for the 60 mg fasted , 60 mg fed , and 75 mg fasted dose levels were 21.3 hours (6.0-54.6), 19.6 hours (7.7-30.0), and 29.2 hours (15.0-88.2), respectively. Mean systemic exposure to paxalisib at fasted state based on AUC0-∞ increased in a manner that was slightly less than dose proportional between 60 mg and 75 mg dose levels. This is probably indicative of a saturation effect at the 75 mg dose. Systemic exposure to paxalisib increased for the 60 mg fed status compared to 60 mg fasted status. Geometric mean ratios for Cmax, AUClast, and AUC0-∞ were 1.40, 1.38, and 1.12, respectively. Conclusion: The pharmacokinetics of paxalisib in newly-diagnosed patients are similar to those in recurrent patients, and remain compatible with daily oral dosing. There is evidence that PK parameters begin to show saturation at doses higher than 60 mg, likely representing the limit of solubility of the molecule. Paxalisib demonstrates a mild food effect, which is unlikely to necessitate fasted administration in clinical practice. These data inform further ongoing studies with paxalisib and have just commenced recruitment into the pivotal GBM AGILE study (NCT03970447). Citation Format: Patrick Y. Wen, John de Groot, James D. Battiste, Samuel A. Goldlust, Denise Damek, James S. Garner, Jeremy A. Simpson, Alan Olivero, Timothy Cloughesy. Pharmacokinetics of paxalisib in phase 2 clinical study in glioblastoma (GBM) with unmethylated O6-methylguanine-methyltransferase (MGMT) promotor status [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB125.

Absorption, metabolism and excretion of pictilisib, a potent Pan-Class I phosphatidylinositol-3-Kinase (PI3K) inhibitor, in rats, dogs, and humans

1. The absorption, metabolism and excretion of pictilisib, a selective small molecule inhibitor of class 1 A phosphoinositide 3-kinase (PI3K), was characterized following a single oral administration of [14C]pictilisib in rats, dogs and humans at the target doses of 30 mg/kg, 5 mg/kg and 60 mg, respectively. 2. Pictilisib was rapidly absorbed with Tmax less than 2 h across species. In systemic circulation, pictilisib represented the predominant total radioactivity greater than 86.6% in all species. 3. Total pictilisib and related radioactivity was recovered from urine and faeces in rats, dogs, and human at 98%, 80% and 95%, respectively, with less than 2% excreted in urine and the rest excreted into faeces. 4. In rat and dog, more than 40% of drug-related radioactivity was excreted into the bile suggesting biliary excretion was the major route of excretion. Unchanged pictilisib was a minor component in rat and dog bile. The major metabolite in bile was O-glucuronide of oxidation on indazole moiety (M20, 21% of the dose) in rats and an oxidative piperazinyl ring-opened metabolite M7 (10.8% of the dose) in dogs. 5. Oxidative glutathione (GSH) conjugates (M18, M19) were novel metabolites detected in rat bile, suggesting the potential generation of reactive intermediates from pictilisib. The structure of M18 was further confirmed by NMR to be a N-hydroxylated and GSH conjugated metabolite on the moiety of the indazole ring.

Structural Insight into the Two-Step Mechanism of PAI-1 Inhibition by Small Molecule TM5484.

Plasminogen activator inhibitor-1 (PAI-1), a key regulator of the fibrinolytic system, is the main physiological inhibitor of plasminogen activators. By interacting with matrix components, including vitronectin (Vn), PAI-1 plays a regulatory role in tissue remodeling, cell migration, and intracellular signaling. Emerging evidence points to a role for PAI-1 in various pathological conditions, including cardiovascular diseases, cancer, and fibrosis. Targeting PAI-1 is therefore a promising therapeutic strategy in PAI-1-related pathologies. A class of small molecule inhibitors including TM5441 and TM5484, designed to bind the cleft in the central β-sheet A of PAI-1, showed to be potent PAI-1 inhibitors in vivo. However, their binding site has not yet been confirmed. Here, we report two X-ray crystallographic structures of PAI-1 in complex with TM5484. The structures revealed a binding site at the flexible joint region, which is distinct from the presumed binding site. Based on the structural analysis and biochemical data we propose a mechanism for the observed dose-dependent two-step mechanism of PAI-1 inhibition. By binding to the flexible joint region in PAI-1, TM5484 might restrict the structural flexibility of this region, thereby inducing a substrate form of PAI-1 followed by a conversion to an inert form.