Isoform-selective Inhibitors Research Articles

Inhibition of K-Ras4B-plasma membrane association with a membrane microdomain-targeting peptide.

The association of K-Ras4B protein with plasma membrane (PM) is required for its signaling activity. Thus, direct inhibition of K-Ras4B–PM interaction could be a potential anti-Ras therapeutic strategy. However, it remains challenging to modulate such protein–PM interaction. Based on Ras isoform-specific PM microdomain localization patterns, we have developed a potent and isoform-selective peptide inhibitor, Memrasin, for detachment of K-Ras4B from the PM. Memrasin is one of the first direct inhibitors of K-Ras4B–PM interaction, and consists of a membrane ld region-binding sequence derived from the C-terminal region of K-Ras4B and an endosome-escape enhancing motif that can aggregate on membrane. It forms peptide-enriched domains in the ld region, abrogates the tethering of K-Ras4B to the PM and accordingly impairs Ras signaling activity, thereby efficiently decreasing the viability of several human lung cancer cells in a dose-responsive and K-Ras dependent manner. Memrasin provides a useful tool for exploring the biological function of K-Ras4B on or off the PM and a potential starting point for further development into anti-Ras therapeutics.

The antibiotic furagin and its derivatives are isoform-selective human carbonic anhydrase inhibitors

The clinically used antibiotic Furagin and its derivatives possess inhibitory activity on human (h) carbonic anhydrases (CA, EC 4.2.1.1), some of which are highly expressed in various tissues and malignancies (hCA IX/XII). Furagin exhibited good hCA IX and XII inhibition with K Is of 260 and 57 nM, respectively. It does not inhibit off-target CA I and poorly inhibited CA II (K I = 9.6 μM). Some synthesised Furagin derivatives with aminohydantoin moieties as zinc binding group exhibited weak inhibition of CA I/II, and good inhibition of CA IX/XII with K Is ranging from 350 to 7400 and 150 to 5600 nM, respectively. Docking and molecular dynamics simulations suggest that selectivity for the cancer-associated CA IX/XII over CA II is due to strong H-bond interactions in CA IX/XII, involving the tail orientated towards hydrophobic area of the active site. These results suggest a possible drug repurposing of Furagin as anti-cancer agent.

PI3K Isoform-Selective Inhibitors in Cancer.

PI3K inhibitors are a common area of research in finding a successful treatment of cancer. The PI3K pathway is important for cell growth, apoptosis, cell metabolism, cell survival, and a multitude of other functions. There are multiple isoforms of PI3K that can be broken down into three categories: class I, II, and III. Each isoform has at least one subunit that helps with the functionality of the isoform. Mutations found in the PI3K isoforms are commonly seen in many different types of cancer and the use of inhibitors is being tested to stop the cell survival of cancer cells. Individual PI3K inhibitors have shown some inhibition of the pathway; however, there is room for improvement. To better treat cancer, PI3K inhibitors are being combined with other pathway inhibitors. These combination therapies have shown better results with cancer treatments. Both the monotherapy and dual therapy treatments are still currently being studied and data collected to better understand cancer and other treatment options.

Isoform-selective NADPH oxidase inhibitor panel for pharmacological target validation

Dysfunctional reactive oxygen species (ROS) signaling is considered an important disease mechanism. Therapeutically, non-selective scavenging of ROS by antioxidants, however, has failed in multiple clinical trials to provide patient benefit. Instead, pharmacological modulation of disease-relevant, enzymatic sources of ROS appears to be an alternative, more promising and meanwhile successfully validated approach. With respect to targets, the family of NADPH oxidases (NOX) stands out as main and dedicated ROS sources. Validation of the different NOX isoforms has been mainly through genetically modified rodent models and is lagging behind in other species. It is unclear whether the different NOX isoforms are sufficiently distinct to allow selective pharmacological modulation. Here we show for five widely used NOX inhibitors that isoform selectivity can be achieved, although individual compound specificity is as yet insufficient. NOX1 was most potently (IC50) targeted by ML171 (0.1 μM); NOX2, by VAS2870 (0.7 μM); NOX4, by M13 (0.01 μM) and NOX5, by ML090 (0.01 μM). In addition, some non-specific antioxidant and assay artefacts may limit the interpretation of data, which included, surprisingly, the clinically advanced NOX inhibitor, GKT136901. In a human ischemic blood-brain barrier hyperpermeability model where genetic target validation is not an option, we provide proof-of-principle that pharmacological target validation for different NOX isoforms is possible by applying an inhibitor panel at IC50 concentrations. Moreover, our findings encourage further lead optimization and development efforts for isoform-selective NOX inhibitors in different indications.

Torin2 Exploits Replication and Checkpoint Vulnerabilities to Cause Death of PI3K-Activated Triple-Negative Breast Cancer Cells.

Frequent mutation of PI3K/AKT/mTOR signaling pathway genes in human cancers has stimulated large investments in targeted drugs but clinical successes are rare. As a result, many cancers with high PI3K pathway activity, such as triple-negative breast cancer (TNBC), are treated primarily with chemotherapy. By systematically analyzing responses of TNBC cells to a diverse collection of PI3K pathway inhibitors, we find that one drug, Torin2, is unusually effective because it inhibits both mTOR and other PI3K-like kinases (PIKKs). In contrast to mTOR-selective inhibitors, Torin2 exploits dependencies on several kinases for S-phase progression and cell-cycle checkpoints, thereby causing accumulation of single-stranded DNA and death by replication catastrophe or mitotic failure. Thus, Torin2 and its chemical analogs represent a mechanistically distinct class of PI3K pathway inhibitors that are uniquely cytotoxic to TNBC cells. This insight could be translated therapeutically by further developing Torin2 analogs or combinations of existing mTOR and PIKK inhibitors.

Histone Deacetylase Inhibitors and Antioxidants From the Root of Gluta usitata

A new glycoside, glutacoside (1), as well as 6 known compounds was isolated and identified from the root of Gluta usitata. Their structures were determined by Infrared spectroscopy, Mass spectroscopy, and 1-Dimensional and 2-dimensional nuclear magnetic resonance spectroscopy data. The histone deacetylase (HDAC) inhibitory and antioxidant activities of the obtained compounds were evaluated. Molecular docking experiments of the selected compound with representatives of class I (HDAC2 and HDAC8) and class II (HDAC4 and HDAC7) HDAC isoforms displayed potential isoform-selective HDAC inhibitors. Molecular docking data showed consistent results to the in vitro experiments with the highest potency against HDAC8. The docking studies suggested that the phenolic and carbonyl group can be favorably accommodated at the gorge region of the binding site. Furthermore, the phenolic groups also acted as major roles for antioxidant activities.

Roles of glycogen synthase kinase 3 alpha and calcineurin in regulating the ability of sperm to fertilize eggs

Glycogen synthase kinase 3 (GSK3) was identified as an enzyme regulating sperm protein phosphatase. The GSK3α paralog, but not GSK3β, is essential for sperm function. Sperm lacking GSK3α display altered motility and are unable to undergo hyperactivation, which is essential for fertilization. Male mice lacking sperm-specific calcineurin (PP2B), a calcium regulated phosphatase, in testis and sperm, are also infertile. Loss of PP2B results in impaired epididymal sperm maturation and motility. The phenotypes of GSK3α and PP2B knockout mice are similar, prompting us to examine the interrelationship between these two enzymes in sperm. High calcium levels must exist to permit catalytically active calcineurin to function during epididymal sperm maturation. Total and free calcium levels are high in immotile compared to motile epididymal sperm. Inhibition of calcineurin by FK506 results in an increase in the net phosphorylation and a consequent decrease in catalytic activity of sperm GSK3. The inhibitor FK506 and an isoform-selective inhibitor of GSK3α, BRD0705, also inhibited fertilization of eggs in vitro. Interrelated functions of GSK3α and sperm PP2B are essential during epididymal sperm maturation and during fertilization. Our results should enable the development of male contraceptives targeting one or both enzymes.

Development of classification models for identification of important structural features of isoform-selective histone deacetylase inhibitors (class I).

As one of the hot topics in the epigenetic studies, histone deacetylases inhibitors (HDACIs) have been introduced to treat a variety of diseases such as cancer, immune disorder and neuronal diseases. Given the high numbers of available pathways in which HDACs are involved, the HDACIs that act particularly on Class I or Class II enzymes are considered as possible candidates for anticancer drugs. Due to their effective roles in the onset of cancer and its progression, HDAC Class I isoforms (HDAC 1, 2, 3 and 8) were considered in this study. Herein, our objective is to determine the important isoform-selective and isoform-active structural features of HDACIs using the valid classification models. For this purpose, a diverse dataset comprising 8224 HDAC modulators was collected from the binding database. To identify the significant discriminative features, five classification models were generated by supervised Kohonen network and support vector machine methods. Variable importance in projection method was used as a variable selection approach. The results obtained from descriptor analysis show that physicochemical properties, such as hydrogen bonding, number of branches, size, flexibility, polarity and sphericity in the structure of molecules, were closely related to the bioactivity of HDACIs. The reliability and predictive ability of the conducted models were evaluated using the tenfold cross-validation techniques, test sets and applicability domain analysis. All of the obtained classification models represented high statistical quality and predictive ability with accuracy greater than 85% for the test sets. The proposed strategy and the selective patterns represented in this paper can be applied by researchers in the pharmaceutical sciences who aim to use the same idea for the design of drugs with improved anticancer properties.

PI3Kδ Inhibition Enhances Sensitivity of Primary High-Risk Childhood B-Cell Acute Lymphoblastic Leukemia Cells to Glucocorticoid Chemotherapy

Introduction: Despite excellent 5-year survival for most patients with B-cell acute lymphoblastic leukemia (B-ALL), those with high-risk (HR) B-ALL continue to have inferior outcomes. The prognosis is even worse for patients who respond poorly to chemotherapy, and improvements to current therapy approaches are needed. Glucocorticoids (GCs) are a key component of chemotherapy regimens. Rapid response to GC monotherapy is a well-documented predictor of favorable outcomes, while slow response to GCs correlates with poor outcomes. We hypothesized that enhancing specific GC toxicity to B-ALL cells can improve clinical outcomes for high-risk patients without increasing systemic toxicity. PI3Kδ is a promising target for this purpose. PI3Kδ is a critical component of the B-cell receptor cell-survival pathway; it restrains GC signaling, while GCs repress its expression. PI3Kδ expression is restricted to lymphoid cells, making it an appealing precision target to enhance GC toxicity in lymphoid cells while minimizing risk of increased systemic GC-associated toxicity. We previously demonstrated synergy between dexamethasone (dex) and the FDA-approved isoform-selective PI3Kδ inhibitor idelalisib (idela) in B-ALL cell lines, patient specimens, and patient-derived xenograft models (Kruth et al Blood 2017). In clinical practice, both dex and prednisone (pred) are essential components of childhood ALL chemotherapy. In this study, we sought to elucidate the extent to which idela in combination with dex or pred could induce synergistic killing of HR B-ALL specimens with various genetic backgrounds. Methods: Peripheral blood or bone marrow from patients ages 1-31 years with newly-diagnosed NCI HR or standard-risk (SR) B-ALL at the University of Iowa Stead Family Children's Hospital was obtained after informed consent on an Institutional Review Board-approved research study. Mononuclear cells were isolated from the specimens via Ficoll gradients and cultured in vitro in 384-well plates. Immediately after isolation, cells were treated with serial dilutions of the GCs dex or pred and idela in RPMI1640+10% FBS medium. Cell viability was measured with PrestoBlue (Invitrogen) after 72 hours of treatment. Survival curves were plotted to determine the LD50 of each drug using GraphPad. Dose effects around the LD50 for each drug were analyzed using Compusyn to determine if the drug combination was synergistic (combination index, CI <1), additive (CI=1), or antagonistic (CI >1). Results: As of August 1, 2019, 5 HR B-ALL patient specimens of various genetic alterations have been tested. Two patients were high-risk at diagnosis due to steroid pre-treatment (MAP011) or by NCI risk criteria (age >10 years and/or WBC count >50K/mm3; MAP014). Three patients with initially SR B-ALL were upstaged due to minimal residual disease (MRD) >1% at day 8 in peripheral blood (MAP012) or marrow MRD ≥0.01% at end of induction (MAP009, MAP010). In our in vitro studies, only one sample (MAP014) showed reduced viability with GC alone, while 4 specimens (all except MAP012) responded to idela alone. Most specimens showed a synergistic response to dex or pred in combination with idela with greater reduction in cell viability compared to GC monotherapies. Surprisingly, one specimen (MAP010) had an antagonistic response with pred and idela combination. This near-haploid B-ALL specimen had reduced viability with idela monotherapy, but an antagonistic response with single-agent pred, raising the question of whether GC/idela synergy is influenced by genetic background. Conclusions: In pilot studies, idela sensitized HR B-ALL cells to GC chemotherapy and is a promising strategy for further evaluation in a larger cohort of specimens. Our results highlight the potential impact of genetic heterogeneity within childhood B-ALL upon differential therapeutic responses to standard steroid chemotherapy and demonstrate a potential precision medicine approach to augment GC sensitivity via combination with selective PI3Kδ inhibition. Disclosures Tasian: Gilead Sciences: Research Funding; Aleta Biotherapeutics: Membership on an entity's Board of Directors or advisory committees; Incyte Corportation: Research Funding.

Development of a Pathway-Directed Drug Screen Platform for Cutaneous T Cell Lymphoma Using Patient-Derived Xenograft Models

Cutaneous T cell lymphoma (CTCL) is an orphan disease and represents 3% of non-Hodgkin lymphomas. Mycosis fungoides and Sézary syndrome (MF/SS) are the most common subtypes of CTCL. The development of targeted therapies for orphan diseases is challenging, but particularly so in the case of MF/SS due to the lack of reliable preclinical models for this malignancy, which is limited to the skin at the early stage, but disseminated to lymph nodes and other organs as disease progresses. Here we report a novel patient-derived xenograft (PDX) mouse model of MF/SS that recapitulates the multi-compartmental nature of CTCL and a blood-based genetic biomarker assay for quantitative monitoring of systemic tumor burden in PDX mice. The PDX models were extensively characterized and exhibit cardinal clinical and histologic features of CTCL, including erythematous scaly skin lesions and eventual lymphomatous dissemination to the spleen and other organs, and maintain the molecular characteristics of their clinical counterparts. The malignant T cells harvested from spleen of PDX mice shared identical TCR sequences and immunophenotypes with corresponding MF/SS patient donor, which featured as losing CD7 and CD26 expression in CD4+ T cells. We also developed a quantitative assay of tumor burden in PDX mice by determining the amount of human β-actin cell-free DNA (cfDNA) in the plasma. The cfDNA levels in the plasma were increased in a linear fashion and correlated with the tumor growth post-implantation. To explore the utility of this PDX model for drug testing, we searched for novel agents for CTCL by performing a high-throughput screen of selected available targeted agents and identified phosphatidylinositol 3-kinase (PI3K) as a high value target. A PI3K inhibitor was advanced to our PDX model with favorable results including disease attenuation and survival prolongation. Further experiments using isoform-specific siRNA knockdowns and isoform-selective PI3K inhibitors identified PI3K-δ as the tumor-driving isoform. Additional studies showed synergistic combination of PI3K-δ inhibitors with histone deacetylase (HDAC) inhibitors. The particularly potent combination of copanlisib and panobinostat is proposed for further clinical development. Disclosures McCormick: Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Aduro BioTech,Inc.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other; BridgeBio Pharma,Inc: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Stock;Ownership; Caris Life Sciences: Honoraria, Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo Co., Ltd: Honoraria, Membership on an entity's Board of Directors or advisory committees; NeuroTrials,LLC: Honoraria, Membership on an entity's Board of Directors or advisory committees; Kura Oncology: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead Sciences,Inc.: Research Funding; Leidos Biomedical: Honoraria, Membership on an entity's Board of Directors or advisory committees; Navire: Membership on an entity's Board of Directors or advisory committees, Other: Stock;Ownership; Riptide Bioscience: Membership on an entity's Board of Directors or advisory committees; Quadriga Biosciences: Membership on an entity's Board of Directors or advisory committees; Portola Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; PMV Pharma: Membership on an entity's Board of Directors or advisory committees; Pfizer,Inc: Honoraria, Membership on an entity's Board of Directors or advisory committees. Ai:Nurix Inc: Research Funding; ADC Therapeutics: Honoraria; Bayer: Honoraria; BMS: Honoraria; Kirin: Honoraria; Immune Design: Honoraria; Seattle Genetics: Honoraria.

Small-Molecule Inhibition of Glucose Transporters GLUT-1-4.

Glucose addiction is observed in cancer and other diseases that are associated with hyperproliferation. The development of compounds that restrict glucose supply and decrease glycolysis has great potential for the development of new therapeutic approaches. Addressing facilitative glucose transporters (GLUTs), which are often upregulated in glucose‐dependent cells, is therefore of particular interest. This article reviews a selection of potent, isoform‐selective GLUT inhibitors and their biological characterization. Potential therapeutic applications of GLUT inhibitors in oncology and other diseases that are linked to glucose addiction are discussed.

Covalent-Allosteric Inhibitors to Achieve Akt Isoform-Selectivity.

Isoforms of protein kinase Akt are involved in essential processes including cell proliferation, survival, and metabolism. However, their individual roles in health and disease have not been thoroughly evaluated. Thus, there is an urgent need for perturbation studies, preferably mediated by highly selective bioactive small molecules. Herein, we present a structure‐guided approach for the design of structurally diverse and pharmacologically beneficial covalent‐allosteric modifiers, which enabled an investigation of the isoform‐specific preferences and the important residues within the allosteric site of the different isoforms. The biochemical, cellular, and structural evaluations revealed interactions responsible for the selective binding profiles. The isoform‐selective covalent‐allosteric Akt inhibitors that emerged from this approach showed a conclusive structure–activity relationship and broke ground in the development of selective probes to delineate the isoform‐specific functions of Akt kinases.

Pharmacokinetics and pharmacodynamics analysis of XQ-1H and its combination therapy with clopidogrel on cerebral ischemic reperfusion injury in rats

Ischemic stroke is the main cause of disability and mortality worldwide. 10-O-(N N-dimethylaminoethyl)-ginkgolide B methane-sulfonate (XQ-1 H) is a novel drug based on the remedial approach for ischemic stroke. Clopidogrel, a widely used anti-platelet drug, is often co-prescribed in the clinic. In this study, we established an UPLC-MS/MS spectrometry method for the determination of XQ-1H and investigated the pharmacokinetic effect of clopidogrel on XQ-1H in rats subjected to middle cerebral artery occlusion (MCAO). Meanwhile, the anti-apoptotic and neuroprotective effects of XQ-1H and its combination with clopidogrel were also studied. The results revealed that XQ-1H and its combination with clopidogrel abridged brain infarct volume, cerebral edema and alleviated neurological dysfunction caused by cerebral ischemic reperfusion injury. Further study demonstrated that XQ-1H combined with clopidogrel lessened TUNEL positive cells, up-regulated bcl-2 expression notably and down-regulated bax expression as compared to both XQ-1H and clopidogrel individually. In addition, a rapid, sensitive UPLC-MS/MS method was developed to quantify the concentration of XQ-1H in MCAO/R rats. Our pharmacokinetic results showed that clopidogrel significantly increased the exposure of XQ-1H, increased the peak plasma concentration (Cmax), area under the curve (AUC) and slowed elimination of XQ-1H in the co-administered group. Besides, for further exploring which CYP450 isoforms are involved in the XQ-1H metabolism, XQ-1H was incubated in human liver microsomes (HLMs) system with or without P450 isoform-selective inhibitors. Our results revealed that clopidogrel altered pharmacokinetics of XQ-1H potentially and subsequently enhanced the pharmacological effect of XQ-1H. Moreover, XQ-1H could be applied as an efficacious neuroprotective agent for ischemic stroke because of its considerable effect on averting neuronal apoptosis.

An isoform-selective inhibitor of tropomyosin receptor kinase A behaves as molecular glue

The production of TrkA-selective inhibitors is considerably difficult because the kinase domains of TrkA and its isoforms TrkB/C have highly homologous amino acid sequences. Here we describe the structural basis for the acquisition of selectivity for a isoform-selective TrkA inhibitor, namely compound V1. The X-ray structure revealed that V1 acts as a molecular glue to stabilize the symmetrical dimer of the TrkA kinase domains. V1 binds to the ATP-binding site and simultaneously engages in the dimeric interface of TrkA. The region of the dimeric interface in TrkA is not conserved in TrkB/C; thus, dimer formation may be a novel mechanism for the production of selective TrkA inhibitors. The biochemical and biophysical assay results confirmed that V1 selectively inhibited TrkA and induced the dimer formation of TrkA, but not TrkB. The binding pocket at the TrkA dimer interface can be used for the production of new isoform-selective TrkA inhibitors.

Multivalent Carbonic Anhydrases Inhibitors.

Biomolecular recognition using a multivalent strategy has been successfully applied, this last decade on several biological targets, especially carbohydrate-processing enzymes, proteases, and phosphorylases. This strategy is based on the fact that multivalent interactions of several inhibitory binding units grafted on a presentation platform may enhance the binding affinity and selectivity. The zinc metalloenzymes carbonic anhydrases (CAs, EC 4.2.1.1) are considered as drug targets for several pathologies, and different inhibitors found clinical applications as diuretics, antiglaucoma agents, anticonvulsants, and anticancer agents/diagnostic tools. Their main drawback is related to the lack of isoform selectivity leading to serious side effects for all pathologies in which they are employed. Thus, the multivalent approach may open new opportunities in the drug design of innovative isoform-selective carbonic anhydrase inhibitors with biomedical applications.

Identification of Isoform-Selective Ligands for the Middle Domain of Heat Shock Protein 90 (Hsp90).

The molecular chaperone heat shock protein 90 (Hsp90) is a current inhibition target for the treatment of diseases, including cancer. In humans, there are two major cytosolic isoforms of Hsp90 (Hsp90α and Hsp90β). Hsp90α is inducible and Hsp90β is constitutively expressed. Most Hsp90 inhibitors are pan-inhibitors that target both cytosolic isoforms of Hsp90. The development of isoform-selective inhibitors of Hsp90 may enable better clinical outcomes. Herein, by using virtual screening and binding studies, we report our work in the identification and characterisation of novel isoform-selective ligands for the middle domain of Hsp90β. Our results pave the way for further development of isoform-selective Hsp90 inhibitors.

Continued exploration and tail approach synthesis of benzenesulfonamides containing triazole and dual triazole moieties as carbonic anhydrase I, II, IV and IX inhibitors

A library of twenty two novel 1,2,3-triazole benzenesulfonamides incorporating thiosemicarbazide, 5(4H)-thione-1,2,4-triazole and variously substituted phenacyl appended 1,2,4-triazole as tail were designed, synthesized and assessed for their efficacy as inhibitors against carbonic anhydrase human (h) isoforms hCA I, II, IV and IX. The physiologically important and off-target cytosolic isoform hCA I was weakly inhibited by most of the newly synthesized sulfonamides while the glaucoma associated isoform hCA II was moderately inhibited with KIs spanning in low nanomolar range (KI = 8.0 nM–0.903 μM). The membrane bound isoform hCA IV, which is known to be involved in glaucoma and retinitis pigmentosa among others, was strongly inhibited by all newly synthesized sulfonamides out of which nine compounds inhibited isoform hCA IV even more effectively as compared to standard drug acetazolamide (AAZ). The membrane bound isoform hCA IX, associated with growth of tumor cells, was moderately inhibited with KIs ranging between 51 nM–3.198 μM. The effect of appending variously substituted tails on heterocyclic moieties over inhibition potential of synthesized sulfonamides is also disclosed which can be of further interest in pharmacological studies for exploring synthesis of isoform selective inhibitors.

PI3K Signaling in Chronic Obstructive Pulmonary Disease: Mechanisms, Targets, and Therapy.

Chronic Obstructive Pulmonary Disease (COPD) is a progressive respiratory disorder characterized by irreversible chronic inflammation and airflow obstruction. It affects more than 64 million patients worldwide and it is predicted to become the third cause of death in the industrialized world by 2030. Currently available therapies are not able to block disease progression and to reduce mortality, underlying the need for a better understanding of COPD pathophysiological mechanisms to identify new molecular therapeutic targets. Recent studies demonstrated that phosphoinositide 3-kinase (PI3K) signaling is prominently activated in COPD and correlates with an increased susceptibility of patients to lung infections. PI3Ks have thus emerged as promising alternative drug targets for COPD and a wide array of pan-isoform and isoform-selective inhibitors have been tested in preclinical models and are currently being evaluated in clinical studies. Here, we summarize the recent knowledge on the involvement of PI3K enzymes in the pathophysiology of COPD, and we discuss the most recent results arising from the preclinical as well as the clinical testing of PI3K inhibitors as novel therapeutics for COPD.

Synthesis and characterization of novel isoform-selective IP6K1 inhibitors.

Inositol hexakisphosphate kinases (IP6Ks) have been increasingly studied as therapeutically interesting enzymes. IP6K isoform specific knock-outs have been used to successfully explore inositol pyrophosphate physiology and related pathologies. A pan-IP6K inhibitor, N2-(m-trifluorobenzyl)-N6-(p-nitrobenzyl) purine (TNP), has been used to confirm phenotypes observed in genetic knock-out experiments; however, it suffers by having modest potency and poor solubility making it difficult to handle for in vitro applications in the absence of DMSO. Moreover, TNP's pan-IP6K inhibitory profile does not inform which IP6K isoform is responsible for which phenotypes. In this report we describe a series of purine-based isoform specific IP6K1 inhibitors. The lead compound was identified after multiple rounds of SAR and has been found to selectively inhibit IP6K1 over IP6K2 or IP6K3 using biochemical and biophysical approaches. It also boasts increased solubility and IP6K1 potency over TNP. These new compounds are useful tools for additional assay development and exploration of IP6K1 specific biology.

Development and Validation of a Higher-Throughput Cytochrome P450 Inhibition Assay with the Novel Cofactor-Supplemented Permeabilized Cryopreserved Human Hepatocytes (MetMax Human Hepatocytes).

Here, we report the application of a novel hepatocyte system, the cofactor-supplemented permeabilized cryopreserved human hepatocytes [MetMax human hepatocytes (MMHHs)] in a higher-throughput 384-well plate assay for the evaluation of cytochrome P450 (P450) inhibition. The assay was created to develop physiologically relevant P450 inhibition information, taking advantage of the complete organelle composition and their associated drug-metabolizing enzymes of the MMHH but with the ease of use of human liver microsomes, including storage at -80°C instead of in liquid nitrogen, and thaw and use without centrifugation and microscopic evaluation as required for intact hepatocytes. Nine key P450 isoforms for drug metabolism (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4) were evaluated using multiple isoform-selective inhibitors. Results with MMHH were found to be comparable to those obtained with intact cryopreserved human hepatocytes (CHHs). Isoform-selective drug-metabolizing enzyme pathways evaluated were phenacetin O-deethylation (CYP1A2), coumarin 7-hydroxylation (CYP2A6), bupropion hydroxylation (CYP2B6), amodiaquine N-deethylation (CYP2C8), diclofenac 4-hydroxylation (CYP2C9), s-mephenytoin 4'-hydroxylation (CYP2C19), dextromethorphan O-demethylation (CYP2D6), chlorzoxazone 6-hydroxylation (CYP2E1), and midazolam 1'-hydroxylation and testosterone 6β-hydroxylation (CYP3A4). The Km values obtained with MMHHs were comparable with those reported in the literature for CHHs. Using substrate concentrations at or near Km values, the IC50 values for the standard inhibitors against the P450 activities were found to be comparable between MMHHs and CHHs, with 73% and 84% of values falling within 2-fold and 3-fold, respectively, from the line of unity. The results indicate that MMHHs can be an efficient experimental system for the evaluation of P450 inhibition in hepatocytes. SIGNIFICANCE STATEMENT: MetMax human hepatocytes (MMHHs) are cofactor-supplemented cryopreserved human hepatocytes with the complete drug-metabolizing enzyme pathways of the conventional hepatocytes but with the convenience of human liver microsomes, including storage at -80°C instead of in liquid nitrogen, and direct thaw and use without a need for centrifugation and microscopic examination. Here, we report the application of MMHH in a high-throughput assay in a 384-well plate format for the evaluation of cytochrome P450 (P450) inhibition. Our results show that data obtained with MMHH are similar to those with conventional hepatocytes, suggesting that the MMHH 384-well P450 inhibition assay can be used routinely for the evaluation of drug-drug interaction potential of new chemical entities in drug development.