Isoform-specific Inhibitors Research Articles

Structural basis for isoform-specific inhibition of human CTPS1

Cytidine triphosphate synthase 1 (CTPS1) is necessary for an effective immune response, as revealed by severe immunodeficiency in CTPS1-deficient individuals [E. Martin et al], [Nature] [510], [288-292] ([2014]). CTPS1 expression is up-regulated in activated lymphocytes to expand CTP pools [E. Martin et al], [Nature] [510], [288-292] ([2014]), satisfying increased demand for nucleic acid and lipid synthesis [L. D. Fairbanks, M. Bofill, K. Ruckemann, H. A. Simmonds], [J. Biol. Chem. ] [270], [29682-29689] ([1995]). Demand for CTP in other tissues is met by the CTPS2 isoform and nucleoside salvage pathways [E. Martin et al], [Nature] [510], [288-292] ([2014]). Selective inhibition of the proliferative CTPS1 isoform is therefore desirable in the treatment of immune disorders and lymphocyte cancers, but little is known about differences in regulation of the isoforms or mechanisms of known inhibitors. We show that CTP regulates both isoforms by binding in two sites that clash with substrates. CTPS1 is less sensitive to CTP feedback inhibition, consistent with its role in increasing CTP levels in proliferation. We also characterize recently reported small-molecule inhibitors, both CTPS1 selective and nonselective. Cryo-electron microscopy (cryo-EM) structures reveal these inhibitors mimic CTP binding in one inhibitory site, where a single amino acid substitution explains selectivity for CTPS1. The inhibitors bind to CTPS assembled into large-scale filaments, which for CTPS1 normally represents a hyperactive form of the enzyme [E. M. Lynch et al], [Nat. Struct. Mol. Biol.] [24], [507-514] ([2017]). This highlights the utility of cryo-EM in drug discovery, particularly for cases in which targets form large multimeric assemblies not amenable to structure determination by other techniques. Both inhibitors also inhibit the proliferation of human primary T cells. The mechanisms of selective inhibition of CTPS1 lay the foundation for the design of immunosuppressive therapies.

Development of PI3K inhibitors: Advances in clinical trials and new strategies (Review)

Phosphatidylinositol 3-kinases (PI3Ks) are the family of vital lipid kinases widely distributed in mammalian cells. The overexpression of PI3Ks leads to hyperactivation of the PI3K/AKT/mTOR pathway, which is considered a pivotal pathway in the occurrence and development of tumors. Hence, PI3Ks are viewed as promising therapeutic targets for anti-cancer therapy. To date, some PI3K inhibitors have achieved desired therapeutic effect via inhibiting the activity of PI3Ks or reducing the level of PI3Ks in clinical trials, among which, Idelalisib, Alpelisib and Duvelisib have been approved by the FDA for treatment of ER+/HER2- advanced metastatic breast cancer and refractory chronic lymphocytic leukemia (CLL) and small lymphocytic lymphomas (SLL). This review focuses on the latest advances of PI3K inhibitors with efficacious anticancer activity, which are classified into Pan-PI3K inhibitors, isoform-specific PI3K inhibitors and dual PI3K/mTOR inhibitors based on the isoform affinity. Their corresponding structure characteristics and structures-activity relationship (SAR), together with the progress in the clinical application are mainly discussed. Additionally, the new PI3K inhibitory strategy, such as PI3K degradation agent, for the design of potential PI3K candidates to overcome drug resistance is referred as well.

Caffeine-induced inversion of prostaglandin E2 effects on hepatic stellate cell activation

Background and aimsLiver inflammation leads to the activation of hepatic stellate cells (HSCs), resulting in the development of liver fibrosis. The present study aimed to investigate the effects of prostaglandin E2 (PGE2), which is biosynthesized by Kupffer cells, hepatocytes, and HSCs during inflammation, on HSC activation, including its combinatory effect with caffeine. MethodsHSCs isolated from mice were activated by culturing in a medium supplemented with 10% fetal bovine serum for 7 days on plastic plates. The activation of HSCs was evaluated by immunofluorescence of α-smooth muscle actin in HSCs. Comprehensive gene expression analysis was performed using mRNA-sequencing to compare HSCs cultured for 1 or 7 days, with or without PGE2, caffeine, or both. ResultsPGE2 (1 μM) facilitated the activation of HSCs but inhibited the HSC activation in the presence of caffeine (3 mM). Comprehensive gene expression analysis revealed that HSCs treated with PGE2 in the presence of caffeine were classified in the same class as HSCs cultured for 1 day, i.e., quiescent HSCs. In contrast, PGE2 did not exhibit an inhibitory effect on HSC activation when co-treated with any isoform-specific phosphodiesterase inhibitors. Although the adenylate cyclase inhibitor 2′,5′-dideoxyadenosine suppressed the elevation of intracellular cAMP level induced by PGE2 in the presence of caffeine, it had no effect on the inhibition of HSC activation by PGE2 plus caffeine. ConclusionThe effect of PGE2 on HSC activation is changed from facilitatory to inhibitory when combined with caffeine, suggesting that caffeine may effectively suppress liver fibrosis during inflammation.

ROCK2-Specific Inhibitor KD025 Suppresses Adipocyte Differentiation by Inhibiting Casein Kinase 2.

KD025, a ROCK2 isoform-specific inhibitor, has an anti-adipogenic activity which is not mediated by ROCK2 inhibition. To identify the target, we searched binding targets of KD025 by using the KINOMEscanTM screening platform, and we identified casein kinase 2 (CK2) as a novel target. KD025 showed comparable binding affinity to CK2α (Kd = 128 nM). By contrast, CK2 inhibitor CX-4945 and ROCK inhibitor fasudil did not show such cross-reactivity. In addition, KD025 effectively inhibited CK2 at a nanomolar concentration (IC50 = 50 nM). We examined if the inhibitory effect of KD025 on adipocyte differentiation is through the inhibition of CK2. Both CX-4945 and KD025 suppressed the generation of lipid droplets and the expression of proadipogenic genes Pparg and Cebpa in 3T3-L1 cells during adipocyte differentiation. Fasudil exerted no significant effect on the quantity of lipid droplets, but another ROCK inhibitor Y-27632 increased the expression of Pparg and Cebpa. Both CX-4945 and KD025 acted specifically in the middle stage (days 1–3) but were ineffective when treated at days 0–1 or the late stages, indicating that CX-4945 and KD025 may regulate the same target, CK2. The mRNA and protein levels of CK2α and CK2β generally decreased in 3T3-L1 cells at day 2 but recovered thereafter. Other well-known CK2 inhibitors DMAT and quinalizarin inhibited effectively the differentiation of 3T3-L1 cells. Taken together, the results of this study confirmed that KD025 inhibits ROCK2 and CK2, and that the inhibitory effect on adipocyte differentiation is through the inhibition of CK2.

EGFRisopred: a machine learning-based classification model for identifying isoform-specific inhibitors against EGFR and HER2.

The EGFR kinase pathway is one of the most frequently activated signaling pathways in human cancers. EGFR and HER2 are the two significant members of this pathway, which are attractive drug targets of clinical relevance in lung and breast cancer. Therefore, identifying EGFR- and HER2-specific inhibitors is one of the important challenges in cancer drug discovery. To address this issue, a dataset of 519 compounds having inhibitory activity against both the isoforms, i.e., EGFR and HER2, was collected from the literature and developed a knowledge-based computational classification model for predicting the specificity of a molecule for an isoform (EGFR/HER2) with precision. A total of seventy-two classification models using nine fingerprint types, four classifiers (IBK, NB, SMO and RF) and two different datasets (EGFR and HER2 isoform specific) were developed. It was observed that the models developed using random forest and IBK performed better for EGFR- and HER2-specific datasets, respectively. Scaffold and functional group analysis led to the identification of prevalent core and fragments in each of the datasets. The accuracy of the selected best performing models was also evaluated using the decoy dataset. We have also developed an application EGFRisopred, which integrates the best performing models and permits the user to predict the specificity of a compound as an EGFR-/HER2-specific anticancer agent. It is expected that the tool's availability as a free utility will allow researchers to identify new inhibitors against these targets important in cancer.

The Emerging Role of the Fetal Insulin Receptor in Hormone-refractory Breast Cancer.

Type 1 insulin-like growth factor receptor (IGF-1R) is a transmembrane tyrosine kinase receptor and a mediator of the biologic effects of insulin-like growth factor (IGF)-I and -II. Inhibitors of IGF-1R signaling were tested in clinical cancer trials aiming to assess the utility of this receptor as a therapeutic target; essentially all IGF-1R inhibitors failed to provide an additional benefit compared with standard-of-care therapy. In this review, we will evaluate the role the insulin receptor (IR) plays in mediating IGF signaling and subsequent metabolic and mitogenic effects as 1 possible reason for these failures. IR is expressed as 2 isoforms, with the fetal isoform IR-A derived from alternative splicing and loss of exon 11, the adult isoform (IR-B) includes this exon. Cancer frequently re-expresses fetal proteins and this appears to be the case in cancer with a re-expression of the fetal isoform and an increased IR-A:IR-B ratio. The biological effects of IR isoform signaling are complex and not completely understood although it has been suggested that IR-A could stimulate mitogenic signaling pathways, play a role in cancer cell stemness, and mediate tolerance to cancer therapies. From a clinical perspective, the IR-A overexpression in cancer may explain why targeting IGF-1R alone was not successful. However, given the predominance of IR-A expression in cancer, it may also be possible to develop isoform specific inhibitors and avoid the metabolic consequences of inhibiting IR-B. If such inhibitors could be developed, then IR-A expression could serve as a predictive biomarker, and cotargeting IR-A and IGF-1R could provide a novel, more effective therapy method.

Abstract 2418: PTEN regulated PI3K-p110 and AKT isoform plasticity controls metastatic prostate cancer progression

Abstract Background: The loss of PTEN function, one of the most frequent mutations in prostate cancer (CaP), is presumed to drive disease progression through AKT activation. However, two transgenic mouse CaP models, Pten/RbPE:-/- and Akap12-/-;RbPE:-/-, exhibited different CaP and metastasis development even though both shared Rb loss plus Akt activation, based on increased relative AKTpoS473 levels. Experimental Procedures: We analyzed whether PI3K-p110 and/or AKT isoform reliance, using isoform-specific RNAi or small molecule inhibitors, played a role in the different CaP outcomes. Findings: In comparison to Akap12/Rb-null high-grade prostatic intraepithelial neoplasias, Pten/Rb-null adenocarcinomas had increased protein and activation levels of AKT2 and decreased relative levels of Smad4, a known CaP metastasis suppressor. Comparison of isogenic PTEN +/- pairs of human or mouse CaP cell lines indicated that PTEN-deficiency correlated with dependence on both p110β and AKT2 for survival or oncogenic growth/invasiveness, whereas PTEN expression correlated with greater dependence on AKT1 plus p110α. Inhibition of AKT2 in PTEN-null CaP cells induced higher SMAD4 levels and decreased chemotaxis and Matrigel invasiveness. Given that PTEN likely regulates non-redundant roles of the AKT isoforms during CaP progression, we sought to characterize AKT isoform substrates by phosphoproteomics analysis. Specifically, after endogenous kinases were inactivated in mouse Pten/Rb-null adenocarcinoma cell lysates using 5'-fluorosulfonylbenzoyl-5'-adenosine (FSBA), dialyzed samples were subjected to in vitro kinase reactions using enzymatically-active, full-length baculovirus-expressed AKT1, AKT2 or AKT3, or as a negative control, a heat-inactivated mixture of all three isoforms. Tryptic peptides isolated on TiO2 beads were subjected to LC-MS/MS. Data will be shown on the role of AKT isoform-shared vs. -preferred substrates in specific parameters of CaP progression controlled by PTEN. Conclusion: Our data suggest that in the context of RB1 loss, PTEN controls a signaling plasticity and drug sensitivity through specific pairings of PI3K-p110 and AKT isoforms. Citation Format: Karina Miller, Seamus Degan, Yanqing Wang, Sheng-Yu Ku, David W. Goodrich, Irwin H. Gelman. PTEN regulated PI3K-p110 and AKT isoform plasticity controls metastatic prostate cancer progression [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 2418.

Blocking HDAC3 in Bone Marrow Stromal Cells Has Direct Anti-Multiple Myeloma Effect and Modulates T Cell Function

IntroductionHistone deacetylases (HDAC) are therapeutic targets in Multiple Myeloma (MM) and the pan-HDAC inhibitor Panobinostat is FDA approved. However, clinical trials showed that non-selective HDAC inhibitors have a narrow therapeutic index, prompting the development of isoform-specific HDAC inhibitors such as the HDAC6 inhibitor Ricolinostat. Preclinical studies have shown that HDAC3 downregulation/inhibition exerts an anti-myeloma effect, suggesting that HDAC3 may be a novel molecular target in MM. However, the effect of targeting HDAC3 in the bone marrow (BM) microenvironment has not been investigated. The BM microenvironment, particularly stromal and immune cells, have been shown to be key in supporting MM growth, immune escape, and resistance to therapy. We sought to study whether targeting HDAC3 in BM stroma cells (BMSC) has direct cytotoxic effects against stromal cells and/or impacts MM cell and T cell function through secreted factors and/or cell-to-cell contact mechanisms.MethodsWe targeted HDAC3 in human BMSC line HS-5 and primary-derived BMSC from MM patients (MM-BMSC) via siRNA or via the HDAC3-selective inhibitor BG45. MTT assay and flow cytometry with annexin V/PI staining were used to assess viability and apoptosis post-HDAC3 KD/pharmacologic inhibition. Downregulation of HDAC3 was confirmed by immunoblotting. To assess the effect of HDAC3 silencing in MM-BMSC on MM cells, we performed siRNA-mediated HDAC3 KD in HS-5 and MM-BMSC, removed transfection media after 48 hours, and performed co-culture experiments with MM1S.Luc and H929.Luc MM cell lines. After 96 hours MM cell proliferation was measured using the Luciferase Assay. Scrambled siRNA transfected HS-5 and MM-BMSC were used as control. To assess the effect of HDAC3 silencing in BMSC on T cells, we isolated peripheral blood mononuclear cells from healthy donors by Ficoll density gradient separation. CD3+ T cells were isolated using EasySep™ Human T Cell Isolation Kit and activated using Dynabeads Human T-Activator CD3/CD28. Cytokine array profiling was performed on the cell-culture supernatant using the Raybiotech C-series Human Cytokine Array according to manufacturer's protocol.ResultsFirst, we showed that pharmacological inhibition of HDAC3 via BG45 does not have direct anti-proliferative or cytotoxic effect on the HS-5 stromal cell line at doses up to 5-fold higher than the EC50 of MM cell lines. Consistent with this data, siRNA-mediated HDAC3 KD does not have direct anti-proliferative and/or cytotoxic effects on HS-5 or MM-BMSC. However, when MM cell lines were co-cultured with HDAC3-silenced HS-5 or MM-BMSC, there was a statistically significant decrease in BMSC-induced MM proliferation compared to control (HS-5: 38% decrease in MM proliferation in HDAC3 KD, p < 0.05; MM-BMSC: 24.1-27.5% decrease in MM proliferation in HDAC3 KD, p < 0.05). To assess whether cell-to-cell contact is necessary to mediate this growth arrest, we cultured MM cell lines in conditioned media from HDAC3 or scrambled siRNA transfected BMSC and discovered that the former was sufficient to induce growth/proliferation arrest (HS-5: 31.5% decrease in MM proliferation in HDAC3 KD, p < 0.05; MM-BMSC: 24.3% decrease in MM proliferation in HDAC3 KD, p < 0.05), suggesting a paracrine effect.We next showed that conditioned supernatant from HDAC3-KD HS-5, cultured alone or in combination with MM cell lines, significantly inhibited the proliferation of activated T-cells (HS-5 supernatant: 47.8% decrease in MM proliferation in HDAC3 KD; HS-5 + MM co-culture supernatant: 32.6% decrease in MM proliferation in HDAC3 KD). To identify the soluble factors responsible for this paracrine effect, we subjected HDAC3-KD and scrambled-transfected HS-5 supernatant to cytokine profiling. We identified a number of cytokines modulated by HDAC3 KD, which we are currently analyzing. Among these, we identified a 1.37-fold increase in soluble PD-L1 when HDAC3 was silenced in HS-5 cells.ConclusionsOur results show that inhibiting HDAC3 in BMSC mediates anti-MM and anti-T cell effect via soluble factors. Gene expression profiling and proteomic studies are currently underway to define the molecular mechanisms supporting these effects. Our preliminary data suggest that targeting HDAC3 in BMSC may alter the checkpoint repertoire in the BM milieu, suggesting potential utility of therapeutic strategies combining HDAC3 and checkpoint inhibitors. DisclosuresHideshima:C4 Therapeutics: Equity Ownership; Acetylon: Consultancy. Mazitschek:Acetylon: Equity Ownership. Anderson:C4 Therapeutics: Other: scientific founder; Oncopep: Other: scientific founder; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; MedImmune: Membership on an entity's Board of Directors or advisory committees; Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees.

Direct P70S6K1 inhibition to replace dexamethasone in synergistic combination with MCL-1 inhibition in multiple myeloma

AbstractNovel combination therapies have markedly improved the lifespan of patients with multiple myeloma (MM), but drug resistance and disease relapse remain major clinical problems. Dexamethasone and other glucocorticoids are a cornerstone of conventional and new combination therapies for MM, although their use is accompanied by serious side effects. We aimed to uncover drug combinations that act in synergy and, as such, allow reduced dosing while remaining effective. Dexamethasone and the myeloid cell leukemia 1 (MCL-1) inhibitor S63845 (MCL-1i) proved the most potent combination in our lethality screen and induced apoptosis of human myeloma cell lines (HMCLs) that was 50% higher compared with an additive drug effect. Kinome analysis of dexamethasone-treated HMCLs revealed a reduction in serine/threonine peptide phosphorylation, which was predicted to result from reduced Akt activity. Biochemical techniques showed no dexamethasone-induced effects on FOXO protein or GSK3 but did show a 50% reduction in P70S6K phosphorylation, downstream of the Akt-mTORC1 axis. Replacing dexamethasone by the P70S6K1 isoform-specific inhibitor PF-4708671 (S6K1i) revealed similar and statistically significant synergistic apoptosis of HMCLs in combination with MCL-1i. Interestingly, apoptosis induced by the P70S6K1i and MCL-1i combination was more-than-additive in all 9 primary MM samples tested; this effect was observed for 6 of 9 samples with the dexamethasone and MCL-1i combination. Toxicity on stem and progenitor cell subsets remained minimal. Combined, our results show a strong rationale for combination treatments using the P70S6K inhibitor in MM. Direct and specific inhibition of P70S6K may also provide a solution for patients ineligible or insensitive to dexamethasone or other glucocorticoids.

Molecular insight into isoform specific inhibition of PI3K-\u03b1 and PKC-\u03b7 with dietary agents through an ensemble pharmacophore and docking studies

Dietary compounds play an important role in the prevention and treatment of many cancers, although their specific molecular mechanism is not yet known. In the present study, thirty dietary agents were analyzed on nine drug targets through in silico studies. However, nine dietary scaffolds, such as silibinin, flavopiridol, oleandrin, ursolic acid, α-boswellic acid, β-boswellic acid, triterpenoid, guggulsterone, and oleanolic acid potentially bound to the cavity of PI3K-α, PKC-η, H-Ras, and Ras with the highest binding energy. Particularly, the compounds silibinin and flavopiridol have been shown to have broad spectrum anticancer activity. Interestingly, flavopiridol was embedded in the pockets of PI3K-α and PKC-η as bound crystal inhibitors in two different conformations and showed significant interactions with ATP binding pocket residues. However, complex-based pharmacophore modeling achieved two vital pharmacophoric features namely, two H-bond acceptors for PI3K-α, while three are hydrophobic, one cat-donor and one H-bond donor and acceptor for PKC-η, respectively. The database screening with the ChemBridge core library explored potential hits on a valid pharmacophore query. Therefore, to optimize perspective lead compounds from the hits, which were subjected to various constraints such as docking, MM/GBVI, Lipinski rule of five, ADMET and toxicity properties. Henceforth, the top ligands were sorted out and examined for vital interactions with key residues, arguably the top three promising lead compounds for PI3K-α, while seven for PKC-η, exhibiting binding energy from − 11.5 to − 8.5 kcal mol−1. Therefore, these scaffolds could be helpful in the development of novel class of effective anticancer agents.

TLR2 dimer-specific ligands selectively activate protein kinase C isoforms in Leishmania infection.

Of the thirteen Toll-like receptors (TLRs) in mice, TLR2 has a unique ability of forming heterodimers with TLR1 and TLR6. Such associations lead to selective cellular signalling and cellular responses such as cytokine expression. One of the signalling intermediates is protein kinase C (PKC); of which, eight isoforms are expressed in macrophages. Leishmania-a protozoan parasite that resides and replicates in macrophages-selectively modulates PKC-α, PKC-β, PKC-δ and PKC-ζ isoforms in macrophages. As TLR2 plays significant roles in Leishmania infection, we examined whether these PKC isoforms play selective roles in TLR2 signalling and TLR2-induced anti-leishmanial functions. We observed that the TLR2 ligands-Pam3 CSK4 (TLR1/2), PGN (TLR2/2) and FSL (TLR2/6)-differentially phosphorylated and translocated PKC-α, PKC-β, PKC-δ and PKC-ζ isoforms to cell membrane in uninfected and L.major-infected macrophages. The PKC isoform-specific inhibitors differentially altered IL-10 and IL-12 expression, Th1 and Th2 responses and anti-leishmanial effects in macrophages and in BALB/c mice. While PKC isoforms' inhibitors had insignificant effects on the Pam3CSK4-induced anti-leishmanial functions, PGN-induced pro-leishmanial effects were enhanced by PKC-(α+β) inhibitors, whereas PKC-(δ+ζ) inhibitors enhanced the anti-leishmanial effects of FSL. These results indicated that the ligand-induced TLR2 dimerization triggered differential dose-dependent and kinetic profiles of PKC isoform activation and that selective targeting of PKC isoforms using their respective inhibitors in combination significantly modulated TLR2-induced anti-leishmanial functions. To the best of our knowledge, this is the first demonstration of TLR2 dimer signalling through PKC isoforms and TLR2-induced PKC isoform-targeted anti-leishmanial therapy.

Structure, biochemistry, and gene expression patterns of the proline biosynthetic enzyme pyrroline-5-carboxylate reductase (PYCR), an emerging cancer therapy target.

Proline metabolism features prominently in the unique metabolism of cancer cells. Proline biosynthetic genes are consistently upregulated in multiple cancers, while the proline catabolic enzyme proline dehydrogenase has dual, context-dependent pro-cancer and pro-apoptotic functions. Furthermore, the cycling of proline and Δ1-pyrroline-5-carboxylate through the proline cycle impacts cellular growth and death pathways by maintaining redox homeostasis between the cytosol and mitochondria. Here we focus on the last enzyme of proline biosynthesis, Δ1-pyrroline-5-carboxylate reductase, known as PYCR in humans. PYCR catalyzes the NAD(P)H-dependent reduction of Δ1-pyrroline-5-carboxylate to proline and forms the reductive half of the proline metabolic cycle. We review the research on the three-dimensional structure, biochemistry, inhibition, and cancer biology of PYCR. To provide a global view of PYCR gene upregulation in cancer, we mined RNA transcript databases to analyze differential gene expression in 28 cancer types. This analysis revealed strong, widespread upregulation of PYCR genes, especially PYCR1. Altogether, the research over the past 20 years makes a compelling case for PYCR as a cancer therapy target. We conclude with a discussion of some of the major challenges for the field, including developing isoform-specific inhibitors, elucidating the function of the long C-terminus of PYCR1/2, and characterizing the interactome of PYCR.

MTOR regulates GPVI-mediated platelet activation

BackgroundDue to mTOR (mammalian/mechanistic target of rapamycin) gene-loss mice die during embryonic development, the role of mTOR in platelets has not been evaluated using gene knockout technology.MethodsA mouse model with megakaryocyte/platelet-specific deletion of mTOR was established, and be used to evaluate the role of mTOR in platelet activation and thrombus formation.ResultsmTOR−/− platelets were deficient in thrombus formation when grown on low-concentration collagen-coated surfaces; however, no deficiency in thrombus formation was observed when mTOR−/− platelets were perfused on higher concentration collagen-coated surfaces. In FeCl3-induced mouse mesenteric arteriole thrombosis models, wild-type (WT) and mTOR−/− mice displayed significantly different responses to low-extent injury with respect to the ratio of occluded mice, especially within the first 40 min. Additionally, mTOR−/− platelets displayed reduced aggregation and dense granule secretion (ATP release) in response to low doses of the glycoprotein VI (GPVI) agonist collagen related peptide (CRP) and the protease-activated receptor-4 (PAR4) agonist GYPGKF-NH2; these deficiencies were overcame by stimulation with higher concentration agonists, suggesting dose dependence of the response. At low doses of GPVI or PAR agonist, the activation of αIIbβ3 in mTOR−/− platelets was reduced. Moreover, stimulation of mTOR−/− platelets with low-dose CRP attenuated the phosphorylation of S6K1, S6 and Akt Ser473, and increased the phosphorylation of PKCδ Thr505 and PKCε Ser729. Using isoform-specific inhibitors of PKCs (δ, ɛ, and α/β), we established that PKCδ/ɛ, and especially PKCδ but not PKCα/β or PKCθ, may be involved in low-dose GPVI-mediated/mTOR-dependent signaling.ConclusionThese observations indicate that mTOR plays an important role in GPVI-dependent platelet activation and thrombus formation.

Growth and Viability of Cutaneous Squamous Cell Carcinoma Cell Lines Display Different Sensitivities to Isoform-Specific Phosphoinositide 3-Kinase Inhibitors.

Cutaneous squamous cell carcinomas (cSCCs) account for about 20% of keratinocyte carcinomas, the most common cancer in the UK. Therapeutic options for cSCC patients who develop metastasis are limited and a better understanding of the biochemical pathways involved in cSCC development/progression is crucial to identify novel therapeutic targets. Evidence indicates that the phosphoinositide 3-kinases (PI3Ks)/Akt pathway plays an important role, in particular in advanced cSCC. Questions remain of whether all four PI3K isoforms able to activate Akt are involved and whether selective inhibition of specific isoform(s) might represent a more targeted strategy. Here we determined the sensitivity of four patient-derived cSCC cell lines to isoform-specific PI3K inhibitors to start investigating their potential therapeutic value in cSCC. Parallel experiments were performed in immortalized keratinocyte cell lines. We observed that pan PI3Ks inhibition reduced the growth/viability of all tested cell lines, confirming the crucial role of this pathway. Selective inhibition of the PI3K isoform p110α reduced growth/viability of keratinocytes and of two cSCC cell lines while affecting the other two only slightly. Importantly, p110α inhibition reduced Akt phosphorylation in all cSCC cell lines. These data indicate that growth and viability of the investigated cSCC cells display differential sensitivity to isoform-specific PI3K inhibitors.

Inhibition of P110α and P110δ catalytic subunits of PI3 kinase reverses impaired arterial healing after injury in hypercholesterolemic male mice.

Endothelial cell (EC) migration is critical for healing arterial injuries, such as those that occur with angioplasty. Impaired re-endothelialization following arterial injury contributes to vessel thrombogenicity, intimal hyperplasia, and restenosis. Oxidized lipid products, including lysophosphatidylcholine (lysoPC), induce canonical transient receptor potential 6 (TRPC6) externalization leading to increased [Ca2+]i, activation of calpains, and alterations of the EC cytoskeletal structure that inhibit migration. The p110α and p110δ catalytic subunit isoforms of phosphatidylinositol 3-kinase (PI3K) regulate lysoPC-induced TRPC6 externalization in vitro. The goal of this study was to assess the in vivo relevance of those in vitro findings to arterial healing following a denuding injury in hypercholesterolemic mice treated with pharmacologic inhibitors of the p110α and p110δ isoforms of PI3K and a general PI3K inhibitor. Pharmacologic inhibition of the p110α or the p110δ isoform of PI3K partially preserves healing in hypercholesterolemic male mice, similar to a general PI3K inhibitor. Interestingly, the p110α, p110δ, and the general PI3K inhibitor do not improve arterial healing after injury in hypercholesterolemic female mice. These results indicate a potential new role for isoform-specific PI3K inhibitors in male patients following arterial injury/intervention. The results also identify significant sex differences in the response to PI3K inhibition in the cardiovascular system, where female sex generally has a cardioprotective effect. This study provides a foundation to investigate the mechanism for the sex differences in response to PI3K inhibition to develop a more generally applicable treatment option.

Histone deacetylase 2: A potential therapeutic target for cancer and neurodegenerative disorders.

Histone deacetylases (HDACs) have been implicated in a number of diseases including cancer, cardiovascular disorders, diabetes mellitus, neurodegenerative disorders and inflammation. For the treatment of epigenetically altered diseases such as cancer, HDAC inhibitors have made a significant progress in terms of development of isoform selective inhibitiors. Isoform specific HDAC inhibitors have less adverse events and better safety profile. A HDAC isoform i.e., HDAC2 demonstrated significant role in the development of variety of diseases, mainly involved in the cancer and neurodegenerative disorders. Discovery and development of selective HDAC2 inhibitors have a great potential for the treatment of target diseases. In the present compilation, we have reviewed the role of HDAC2 in progression of cancer and neurodegenerative disorders, and information on the drug development opportunities for selective HDAC2 inhibition.

Abstract PS11-17: Combination PI3K and NOS targeted therapy for metaplastic breast cancer

Abstract Metaplastic breast cancer (MpBC) is a rare subset accounting for less than 1% of all breast cancers. MpBC exhibits the worst prognosis in comparison to triple-negative breast cancer (TNBC) with a dismal poor survival rate in patients with metastatic disease. The main therapeutic options for MpBC remain surgery and systemic chemotherapy, despite known resistance to most systemic chemotherapy. A common molecular alteration in MpBC is hyperactivation of the phosphoinositide 3-kinase (PI3K) pathway. Recently, we published that MpBC displays a gain-of-function oncogenic mutation in ribosomal protein L39 (RPL39), which is responsible for treatment resistance, stem cell self-renewal, and lung metastasis. The mechanistic function of RPL39 is mediated through inducible nitric oxide synthase (iNOS)-mediated nitric oxide production. In addition, we demonstrated that inhibiting this nitric oxide synthase (NOS) pathway using pan-NOS inhibitor NG-methyl-L-arginine acetate (L-NMMA) may represent a highly effective therapeutic option for MBC patients. Therefore, we hypothesize that the combinatorial approach of inhibiting the two major pathways implicated in MpBC, namely PI3K/Akt/mTOR and NOS pathways would lead to significant tumor regression. Alpelisib, an FDA-approved, isoform-specific PI3K inhibitor, is currently used with the antiestrogen, fulvestrant, to treat hormone receptor (HR)-positive, PIK3CA-mutated breast cancer patients. We used MpBC cell lines Hs578t and BT549 and MpBC patient-derived xenograft (PDX) models in our preliminary studies. Using flow cytometry to detect Annexin V+/DAPI+ cells, we found increased cell death in MpBC cell lines treated with L-NMMA+alpelisib combination treatment in comparison to monotherapy. Immunoblotting of samples from single (L-NMMA or alpelisib) or combination treated cell lines and PDXs showed increased PARP degradation and cleaved caspase 3/9 with combination treatment. In vivo data using PDX models showed that combination treatment of L-NMMA+alpelisib was most effective at reducing tumor volume in comparison to monotherapy. We performed preliminary bulk-RNA sequencing analysis of tumor samples collected from PDX BCM-4664 treated with vehicle control, monotherapy of L-NMMA or alpelisib, and combination therapy. Gene set enrichment analysis found that E2F and Hedgehog signaling pathways were the top two enriched pathways in BCM-4664 tumors treated with combination therapy. Pathway-focused RT-PCR of MpBC PDXs confirmed GSEA results and showed significant gene expression alterations involving E2F signaling and cell cycle regulation. Further studies are currently ongoing to elucidate the molecular mechanisms involved in enhanced cell death and decreased tumorigenesis with L-NMMA and alpelisib dual therapy. Our results support the concept that LNMMA and alpelisib combination therapy has therapeutic potential in the treatment of MpBC, which may enable rapid translational into clinical trials, and impact the exceeding poor prognosis of women with MpBC. Citation Format: Tejaswini Parlapalle Reddy, Roberto R Rosato, Liliana Guzman, Wei Qian, Jianying Zhou, Hongbin Wang, Helen Piwnica-Worms, Stacy Moulder, Jenny C Chang. Combination PI3K and NOS targeted therapy for metaplastic breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS11-17.

Improved Inhibitory and Absorption, Distribution, Metabolism, Excretion, and Toxicology (ADMET) Properties of Blebbistatin Derivatives Indicate That Blebbistatin Scaffold Is Ideal for drug Development Targeting Myosin-2.

Blebbistatin, para-nitroblebbistatin (NBleb), and para-aminoblebbistatin (AmBleb) are highly useful tool compounds as they selectively inhibit the ATPase activity of myosin-2 family proteins. Despite the medical importance of the myosin-2 family as drug targets, chemical optimization has not yet provided a promising lead for drug development because previous structure-activity-relationship studies were limited to a single myosin-2 isoform. Here we evaluated the potential of blebbistatin scaffold for drug development and found that D-ring substitutions can fine-tune isoform specificity, absorption-distribution-metabolism-excretion, and toxicological properties. We defined the inhibitory properties of NBleb and AmBleb on seven different myosin-2 isoforms, which revealed an unexpected potential for isoform specific inhibition. We also found that NBleb metabolizes six times slower than blebbistatin and AmBleb in rats, whereas AmBleb metabolizes two times slower than blebbistatin and NBleb in human, and that AmBleb accumulates in muscle tissues. Moreover, mutagenicity was also greatly reduced in case of AmBleb. These results demonstrate that small substitutions have beneficial functional and pharmacological consequences, which highlight the potential of the blebbistatin scaffold for drug development targeting myosin-2 family proteins and delineate a route for defining the chemical properties of further derivatives to be developed. SIGNIFICANCE STATEMENT: Small substitutions on the blebbistatin scaffold have beneficial functional and pharmacological consequences, highlighting their potential in drug development targeting myosin-2 family proteins.

Identifying requirements for RSK2 specific inhibitors

Identifying isoform-specific inhibitors for closely related kinase family members remains a substantial challenge. The necessity for achieving this specificity is exemplified by the RSK family, downstream effectors of ERK1/2, which have divergent physiological effects. The natural product, SL0101, a flavonoid glycoside, binds specifically to RSK1/2 through a binding pocket generated by an extensive conformational rearrangement within the RSK N-terminal kinase domain (NTKD). In modelling experiments a single amino acid that is divergent in RSK3/4 most likely prevents the required conformational rearrangement necessary for SL0101 binding. Kinetic analysis of RSK2 association with SL0101 and its derivatives identified that regions outside of the NTKD contribute to stable inhibitor binding. An analogue with an n-propyl-carbamate at the 4” position on the rhamnose moiety was identified that forms a highly stable inhibitor complex with RSK2 but not with RSK1. These results identify a SL0101 modification that will aid the identification of RSK2 specific inhibitors.

Influence of stereochemistry on the activity of rapadocin, an isoform-specific inhibitor of the nucleoside transporter ENT1.

Rapadocin is a novel rapamycin-inspired polyketide–tetrapeptide hybrid macrocycle that possesses highly potent and isoform-specific inhibitory activity against the human equilibrative nucleoside transporter 1 (hENT1). Rapadocin contains an epimerizable chiral center in phenylglycine and an olefin group, and can thus exist as a mixture of four stereoisomers. Herein, we report the first total synthesis of the four stereoisomers of rapadocin using two different synthetic strategies and the assignment of their structures. The inhibitory activity of each of the four synthetic isomers on both hENT1 and hENT2 was determined. It was found that the stereochemistry of phenylglycine played a more dominant role than the configuration of the olefin in the activity of rapadocin. These findings will guide the future design and development of rapadocin analogs as new modulators of adenosine signaling.