Alternative Drug Targets Research Articles

Identification and characterization of the Fasciola hepatica sodium- and chloride-dependent taurine transporter.

The parasitic liver fluke Fasciola hepatica infests mainly ruminants, but it can also cause fasciolosis in people, who ingest the metacercariae encysted on plants. The drug of choice to treat fasciolosis is triclabendazole (TBZ), which has been on the market for several decades. This is also true for the other available drugs. Accordingly, drug-resistant flukes have been emerging at an increasing rate making it desirable to identify alternative drug targets. Here, we focused on the fact that adult F. hepatica persists in the hostile environment of the bile ducts of infected organisms. A common way to render bile acids less toxic is to conjugate them to taurine (2-aminoethanesulfonic acid). We cloned a transporter from the solute carrier-6 (SLC6) family, which was most closely related to the GABA-transporter-2 of other organisms. When heterologously expressed, this F. hepatica transporter supported the high-affinity cellular uptake of taurine (KM = 12.0 ± 0.5 μM) but not of GABA. Substrate uptake was dependent on Na+- and Cl- (calculated stoichiometry 2:1). Consistent with the low chloride concentration in mammalian bile, the F. hepatica transporter had a higher apparent affinity for Cl- (EC50 = 14±3 mM) than the human taurine transporter (EC50 = 55±7 mM). We incubated flukes with unconjugated bile acids in the presence and absence of taurine: taurine promoted survival of flukes; the taurine transporter inhibitor guanidinoethansulfonic acid abolished this protective effect of taurine. Based on these observations, we conclude that the taurine transporter is critical for the survival of liver flukes in the bile. Thus, the taurine transporter represents a candidate drug target.

Beyond Channel Activity: Protein-Protein Interactions Involving Viroporins.

Viroporins are short polypeptides encoded by viruses. These small membrane proteins assemble into oligomers that can permeabilize cellular lipid bilayers, disrupting the physiology of the host to the advantage of the virus. Consequently, efforts during the last few decades have been focused towards the discovery of viroporin channel inhibitors, but in general these have not been successful to produce licensed drugs. Viroporins are also involved in viral pathogenesis by engaging in critical interactions with viral proteins, or disrupting normal host cellular pathways through coordinated interactions with host proteins. These protein-protein interactions (PPIs) may become alternative attractive drug targets for the development of antivirals. In this sense, while thus far most antiviral molecules have targeted viral proteins, focus is moving towards targeting host proteins that are essential for virus replication. In principle, this largely would overcome the problem of resistance, with the possibility of using repositioned existing drugs. The precise role of these PPIs, their strain- and host- specificities, and the structural determination of the complexes involved, are areas that will keep the fields of virology and structural biology occupied for years to come. In the present review, we provide an update of the efforts in the characterization of the main PPIs for most viroporins, as well as the role of viroporins in these PPIs interactions.

Targeting BCR-ABL-Independent TKI Resistance in Chronic Myeloid Leukemia by mTOR and Autophagy Inhibition.

BackgroundImatinib and second-generation tyrosine kinase inhibitors (TKIs) nilotinib and dasatinib have statistically significantly improved the life expectancy of chronic myeloid leukemia (CML) patients; however, resistance to TKIs remains a major clinical challenge. Although ponatinib, a third-generation TKI, improves outcomes for patients with BCR-ABL-dependent mechanisms of resistance, including the T315I mutation, a proportion of patients may have or develop BCR-ABL-independent resistance and fail ponatinib treatment. By modeling ponatinib resistance and testing samples from these CML patients, it is hoped that an alternative drug target can be identified and inhibited with a novel compound.MethodsTwo CML cell lines with acquired BCR-ABL-independent resistance were generated following culture in ponatinib. RNA sequencing and gene ontology (GO) enrichment were used to detect aberrant transcriptional response in ponatinib-resistant cells. A validated oncogene drug library was used to identify US Food and Drug Administration–approved drugs with activity against TKI-resistant cells. Validation was performed using bone marrow (BM)–derived cells from TKI-resistant patients (n = 4) and a human xenograft mouse model (n = 4–6 mice per group). All statistical tests were two-sided.ResultsWe show that ponatinib-resistant CML cells can acquire BCR-ABL-independent resistance mediated through alternative activation of mTOR. Following transcriptomic analysis and drug screening, we highlight mTOR inhibition as an alternative therapeutic approach in TKI-resistant CML cells. Additionally, we show that catalytic mTOR inhibitors induce autophagy and demonstrate that genetic or pharmacological inhibition of autophagy sensitizes ponatinib-resistant CML cells to death induced by mTOR inhibition in vitro (% number of colonies of control[SD], NVP-BEZ235 vs NVP-BEZ235+HCQ: 45.0[17.9]% vs 24.0[8.4]%, P = .002) and in vivo (median survival of NVP-BEZ235- vs NVP-BEZ235+HCQ-treated mice: 38.5 days vs 47.0 days, P = .04).ConclusionCombined mTOR and autophagy inhibition may provide an attractive approach to target BCR-ABL-independent mechanism of resistance.

Neuropilin 1 Involvement in Choroidal and Retinal Neovascularisation.

PurposeInhibiting VEGF is the gold standard treatment for neovascular age-related macular degeneration (AMD). It is also effective in preventing retinal oedema and neovascularisation (NV) in diabetic retinopathy (DR) and retinal vein occlusions (RVO). Neuropilin 1 (Nrp1) is a co-receptor for VEGF and many other growth factors, and therefore a possible alternative drug target in intra ocular neovascular disease. Here we assessed choroidal and retinal NV in an inducible, endothelial specific knock out model for Nrp1.MethodsCrossing Nrp1 floxed mice with Pdgfb-CreERT2 mice produced tamoxifen-inducible, endothelial specific Nrp1 knock out mice (Nrp1ΔEC) and Cre-negative, control littermates. Cre-recombinase activity was confirmed in the Ai3(RCL-EYFP) reporter strain. Animals were subjected to laser-induced CNV (532 nm) and spectral domain-optical coherence tomography (SD-OCT) was performed immediately after laser and at day 7. Fluorescein angiography (FA) evaluated leakage and postmortem lectin staining in flat mounted RPE/choroid complexes was also used to measure CNV. Furthermore, retinal neovascularisation in the oxygen induced retinopathy (OIR) model was assessed by immunohistochemistry in retinal flatmounts.ResultsIn vivo FA, OCT and post-mortem lectin staining showed a statistically significant reduction in leakage (p<0.05), CNV volume (p<0.05) and CNV area (p<0.05) in the Nrp1ΔEC mice compared to their Cre-negative littermates. Also the OIR model showed reduced retinal NV in the mutant animals compared to wild types (p<0.001).ConclusionWe have demonstrated reduced choroidal and retinal NV in animals that lack endothelial Nrp1, confirming a role of Nrp1 in those processes. Therefore, Nrp1 may be a promising drug target for neovascular diseases in the eye.

Antifungal Resistance, Metabolic Routes as Drug Targets, and New Antifungal Agents: An Overview about Endemic Dimorphic Fungi.

Diseases caused by fungi can occur in healthy people, but immunocompromised patients are the major risk group for invasive fungal infections. Cases of fungal resistance and the difficulty of treatment make fungal infections a public health problem. This review explores mechanisms used by fungi to promote fungal resistance, such as the mutation or overexpression of drug targets, efflux and degradation systems, and pleiotropic drug responses. Alternative novel drug targets have been investigated; these include metabolic routes used by fungi during infection, such as trehalose and amino acid metabolism and mitochondrial proteins. An overview of new antifungal agents, including nanostructured antifungals, as well as of repositioning approaches is discussed. Studies focusing on the development of vaccines against antifungal diseases have increased in recent years, as these strategies can be applied in combination with antifungal therapy to prevent posttreatment sequelae. Studies focused on the development of a pan-fungal vaccine and antifungal drugs can improve the treatment of immunocompromised patients and reduce treatment costs.

Acalabrutinib Monotherapy in Patients with Ibrutinib Intolerance: Results from the Phase 1/2 ACE-CL-001 Clinical Study

▪Background: Bruton tyrosine kinase (BTK) is a validated target in CLL. Ibrutinib is a BTK inhibitor that has dramatically improved progression-free survival (PFS) and overall survival (OS) of CLL patients (pts) over conventional therapies used in this disease. In a subset of pts receiving ibrutinib, adverse events (AEs), such as diarrhea, rash, myalagias/arthralgias, atrial fibrillation, and bleeding occur and may be related to alternative drug targets, leading to treatment discontinuations or dose reductions. Having an alternative, more selective BTK inhibitor that is tolerable is of high priority for this pt population. Acalabrutinib, a highly selective, potent BTK inhibitor designed to minimize alternative kinase inhibition was evaluated in pts with CLL/SLL in the Phase 1/2 ACE-CL-001 clinical study (NCT02029443).Methods: Eligibility criteria for the ibrutinib-intolerant cohort included confirmed CLL/SLL, intolerance due to ibrutinib-related AEs, Eastern Cooperative Oncology Group performance status ≤2 and age ≥18 years. Pts were treated with acalabrutinib 100 mg twice daily (n=30) or 200 mg daily (n=3) until disease progression or discontinuation for another reason. The primary objective was to determine the safety of acalabrutinib, as assessed by frequency, severity, and attribution of AEs based on CTCAE criteria. The secondary objective was to determine response rate by investigator assessment. Overall response rate (ORR), PFS, and duration of response (DOR) were calculated. Pts were defined as evaluable for response based on ≥1 response assessment after the first dose of study drug, presence of measurable disease, and established modified response criteria (Hallek et al. 2008).Results: Thirty-three pts were treated in the ibrutinib-intolerant cohort. Results are presented through 01 June 2016. Median age was 64 years (range, 50 to 82). Pts were high risk with derived baseline Rai stage III-IV (52%), bulky lymph nodes ≥5 cm (31%), del17p in 8 of 27 patients (30%), del11q in 7 of 27 patients (26%), and unmutated IGHV(81%) at time of study entry. Median duration of prior ibrutinib treatment was 10.5 months (range, 1.0 to 62.3), and median duration from end of ibrutinib to start of acalabrutinib was 47 days (range, 3 to 331). The median follow-up period (n=33) was 9.5 months (range, 0.5 to 20.6). At the time of data cutoff, 24 pts (73%) continued on treatment. Most AEs were Grade (G) 1/2 (58%). The most common AEs (≥15% of pts) were diarrhea (42%), headache (39%), cough (24%), increased weight (24%), nausea (21%), contusion, ecchymosis, fatigue, pyrexia (each in 18%) and myalgia, rash, urinary tract infection and vomiting (each in 15%). Pneumonia (1 G3 and 1 G4) was the only serious AE occurring in ≥2 patients. There were 2 G5 events - a cerebrovascular accident and a fungal infection - both unrelated to study drug. Two cases of treatment-emergent atrial fibrillation occurred: 1 G2 in a pt with a history of paroxysmal atrial fibrillation (G2) on ibrutinib who remains on study and 1 G3 atrial fibrillation in a patient who was able to continue acalabrutinib treatment until coming off study due to disease progression. There were no major hemorrhages, including among the 6 pts who previously developed bleeding events while on ibrutinib. Treatment discontinuations occurred in 9 (27%) patients: progressive disease in 5 (15%), AEs in 2 (6%; 1 unrelated G5 fungal infection and 1 unrelated G3 metastatic endometrial cancer), and 2 (6%) by physician decision. In patients with baseline measurable disease or lymphocytosis, the ORR (CR+PR+PRL) (n=29) was 76%, with 1 CR, 14 PR, and 7 PRL. Median time to PR or better was 3.7 months (95% CI, 1.9 to 5.5). Among 15 responders, median DOR was 13 months (95% CI, 5.6 to 13) at the time of data cutoff. Median PFS has not been reached.Conclusions: Acalabrutinib is well tolerated and effective in most ibrutinib-intolerant pts, providing a potential avenue to continue impactful BTK inhibitor therapy. An ongoing Phase 2 study (NCT02717611) is evaluating acalabrutinib monotherapy in pts with relapsed/refractory CLL and ibrutinib intolerance. DisclosuresAwan:Innate Pharma: Research Funding; Novartis Oncology: Consultancy; Pharmacyclics: Consultancy. Schuh:Novartis: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; GSK: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Gilead: Consultancy, Honoraria. Brown:Infinity: Consultancy; Janssen: Consultancy; Celgene: Consultancy; Roche/Genentech: Consultancy; Pfizer: Consultancy; Gilead: Consultancy; Sun BioPharma: Consultancy; Pharmacyclics: Consultancy; Abbvie: Consultancy. Furman:Genentech: Consultancy; Pharmacyclics: Consultancy, Speakers Bureau; Gilead Sciences: Consultancy; Abbvie: Consultancy, Honoraria; Janssen: Consultancy. Hillmen:Pharmacyclics: Research Funding; Janssen: Honoraria, Research Funding; Roche: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Abbvie: Research Funding. Stephens:Lymphoma Research Foundation: Research Funding. Hamdy:Acerta Pharma: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Huang:Acerta Pharma: Employment. Izumi:Acerta Pharma: Employment, Equity Ownership. Patel:Acerta Pharma: Employment. Wang:Acerta Pharma: Employment, Equity Ownership.

Chronic exposure to cigarette smoke leads to activation of p21 (RAC1)-activated kinase 6 (PAK6) in non-small cell lung cancer cells

Epidemiological data clearly establishes cigarette smoking as one of the major cause for lung cancer worldwide. Recently, targeted therapy has become one of the most preferred modes of treatment for cancer. Though certain targeted therapies such as anti-EGFR are in clinical practice, they have shown limited success in lung cancer patients who are smokers. This demands discovery of alternative drug targets through systematic investigation of cigarette smoke-induced signaling mechanisms. To study the signaling events activated in response to cigarette smoke, we carried out SILAC-based phosphoproteomic analysis of H358 lung cancer cells chronically exposed to cigarette smoke. We identified 1,812 phosphosites, of which 278 phosphosites were hyperphosphorylated (≥ 3-fold) in H358 cells chronically exposed to cigarette smoke. Our data revealed hyperphosphorylation of S560 within the conserved kinase domain of PAK6. Activation of PAK6 is associated with various processes in cancer including metastasis. Mechanistic studies revealed that inhibition of PAK6 led to reduction in cell proliferation, migration and invasion of the cigarette smoke treated cells. Further, siRNA mediated silencing of PAK6 resulted in decreased invasive abilities in a panel of non-small cell lung cancer (NSCLC) cells. Consistently, mice bearing tumor xenograft showed reduced tumor growth upon treatment with PF-3758309 (group II PAK inhibitor). Immunohistochemical analysis revealed overexpression of PAK6 in 66.6% (52/78) of NSCLC cases in tissue microarrays. Taken together, our study indicates that PAK6 is a promising novel therapeutic target for NSCLC, especially in smokers.

Abstract 880: Mapping the metastatic colorectal cancer phospho-proteome for predicting response to cetuximab

Abstract Introduction: The discovery of the key role of the epidermal growth factor receptor (EGFR) and its downstream signaling effectors in the pathophysiology of colorectal cancer (CRC) has resulted in the clinical use of targeted therapies in the treatment of metastatic CRC (mCRC). However, clinical benefit to EGFR blockade is observed in only a subgroup of CRC patients wild type for KRAS andNRAS. Genomic characterization of patient derived xenograft (PDX) models of mCRC have revealed additional resistance mechanisms to the EGFR MAb cetuximab including amplification of MET, HER2 and mutations in ERBB2, EGFR, FGFR1, PDGFRA, and MAP2K1 (Bertotti et al. 2011, 2015). However, not all resistance is understood and the biochemical signaling correlates of sensitivity and resistance remain incompletely understood. Phosphoproteomics allows for comprehensive protein phosphorylation profiling, and thereby may shed new insights into the biology of CRC and the molecular basis of cetuximab response. Aim: To identify aberrant signaling pathways, predictive biomarkers, and alternative drug targets in genomically-annotated CRC PDX models by mass spectrometry-based phosphoproteomics. Methods: We performed LC-MS/MS based phosphoproteome profiling (van der Mijn et al. 2015; Piersma et al. 2015) on untreated CRC PDX models lacking KRAS, NRAS or BRAF mutation (n = 20). For comparison, KRAS, BRAF or NRAS-mutant and MET or HER2 amplified models (n = 21) were analyzed. Phosphopeptides were captured by phosphotyrosine antibodies and titanium dioxide. MS/MS data was searched using MaxQuant. Phosphoproteome data (ion intensities and spectral counts) were correlated with drug sensitivity. Dedicated bioinformatics was used to identify hyperactive kinases and signaling pathways in individual tumors. Results: The dataset contained 13.418 tyrosine-, serine- and threonine-phosphorylated peptides derived from 4588 proteins, including 237 phosphorylated kinases. Unsupervised hierarchical clustering of phosphopeptide intensities revealed subclusters enriched for sensitive and resistant models. Among cetuximab-resistant models, HER2 and MET were identified as hyperphosphorylated and hyperactive driver kinases, confirming the genomic annotation. Sensitivity to combination treatment of both EGFR and driver kinases was observed previously, providing proof-of-principle of the use of phosphoproteomics for the identification of driver kinases. Conclusion/Future: This project is uncovering the phosphoproteomic landscape of colorectal tumors and will yield a better understanding of signaling pathways involved, with an emphasis on identifying activated drug targets and predictive biomarkers in this disease, in relation to KRAS/NRAS/BRAF status and response to anti-EGFR therapy. Citation Format: Robin Beekhof, Mariette Labots, Jaco C. Knol, Tim R.A. Schelfhorst, Nicole van Grieken, Sander R. Piersma, Thang V. Pham, Andrea Bertotti, Livio Trusolino, Henk M.W. Verheul, Connie R. Jiménez. Mapping the metastatic colorectal cancer phospho-proteome for predicting response to cetuximab. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 880.

Abstract 1272: Apelin/Apj pathway for targeting ovarian tumor microenvironment

Abstract Introduction: Ovarian cancer generates unique tumor microenvironment (TME) that promotes and enhances tumor progression and metastasis. Current therapy option for modulating ovarian TME is antiangiogenic therapy but its clinical benefit is limited; thus, alternative drug targets are needed for therapeutic durability. Apelin and its cognate receptor APJ is known to have roles in glucose metabolism, cardiovascular functions, and angiogenesis. We found that apelin/APJ are highly overexpressed in human ovarian tumors, but their functional role is unclear. We also found a significant upregulation of apelin/APJ in tumors that progressed after antiangiogenic therapy in preclinical ovarian cancer models. Our objective is to determine the functional roles and mechanisms of apelin/APJ pathway on promoting ovarian tumor angiogenesis and progression. Methods: We examined autocrine and paracrine functions of apelin/APJ signaling on cell proliferation, migration, and tube formation using human ovarian cancer cells (SKOV3Apln and SKOV3Apj) and endothelial cells (HUVECApj). Phosphoproteome array was performed to evaluate the apelin/APJ downstream signaling. We characterized the effect of apelin or APJ overexpression on tumor development and response to VEGFR inhibitor in vivo. To confirm the clinical relevance of apelin/APJ in ovarian TME, we measured soluble apelin levels in patients’ ascites and APJ expressions in human ovarian tumors using an immunohistochemistry. Results: Apelin (10-100 ng/ml) induced mitogenic and chemoattractant effects on both cancer and endothelial cells. These effects were more prominent under hypoxic condition, reflecting the significance of apelin/APJ axis in TME. The aplein-mediated proliferative and migratory effects were inhibited by a pharmacological APJ inhibitor (ML-221) in a dose-dependent manner. High APJ expression resulted in enhanced pro-angiogenic activity in HUVEC. Overexpression of apelin/APJ also led to reduced response to antiangiogenic treatment in both HUVEC and cancer cells. Apelin signaling induces phosphorylation of AKT, STAT, CREB, PRAS 40, and AMPKα2 in SKOV3 cells. Our xenograft study indicates APJ-overexpressing tumors showed reduced response to sorafenib treatment compared to those control tumors. We found that APJ is mainly expressed in tumors and some extent in stromal component of human ovarian tumors. The median soluble apelin levels in ascites were 187 pg/ml (6.3 - 4,000 pg/ml). Conclusions: Our results suggest that apelin/APJ may play an important role in promoting angiogenesis and progression in ovarian cancer and serve as an attractive pathway targeting ovarian TME. Citation Format: Bharat Kumar Devapatla, Pharavee Jaiprasart, Samrita Dogra, Jihee Ha, Sukyung Woo. Apelin/Apj pathway for targeting ovarian tumor microenvironment. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1272.

SLC6A14 Enhances CFTR Channel Activity in the Cystic Fibrosis Affected Lung Epithelium

Cystic fibrosis (CF) is a common genetic disorder caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. CFTR protein functions apically on epithelial cells as an anion channel, helping to keep the surface hydrated. F508del-mutation in CFTR is the most common CF-causing mutation, which leads to decreased functional expression of the protein. However, patients homozygous for F508del mutation show a large variation in disease severity. To address this variation, previous genome-wide association studies have shown that SLC6A14 is a major modifier of CF disease severity. SLC6A14 is an electrogenic neutral/cationic amino-acid transporter expressed in colonic and lung epithelia. We examined the hypothesis that SLC6A14 has a positive impact on the functional expression of Wt and F508del-CFTR. Consistent with this hypothesis, we found that CFTR mediated anion-flux was enhanced by over-expression of SLC6A14 and addition of its transported substrate: arginine in a heterologous expression system. Similarly, in airway cell cultures endogenously expressing both SLC6A14 and CFTR, we observed that shRNA mediated knockdown of SLC6A14 reduced CFTR channel function. These findings could be translated to primary lung epithelial cultures derived from CF patients. The mechanism underlying the positive effect of SLC6A14 on CFTR function is currently unclear. It unlikely to be mediated via a direct interaction, since the two proteins could not be co-immunoprecipitated. However, it could be due to activation of intracellular signaling pathways leading to change in CFTR channel activity. Future studies will focus on the exploration of these pathways, and whether they affect the phosphorylation status of CFTR. Taken together, these findings give biologic insight to support that SLC6A14 is a modifier of CF disease severity. Hence, it can be used as an alternative drug target, especially in patients resistant to currently available therapies.

Impact of germline and somatic missense variations on drug binding sites

Advancements in next-generation sequencing (NGS) technologies are generating a vast amount of data. This exacerbates the current challenge of translating NGS data into actionable clinical interpretations. We have comprehensively combined germline and somatic nonsynonymous single-nucleotide variations (nsSNVs) that affect drug binding sites in order to investigate their prevalence. The integrated data thus generated in conjunction with exome or whole-genome sequencing can be used to identify patients who may not respond to a specific drug because of alterations in drug binding efficacy due to nsSNVs in the target protein’s gene. To identify the nsSNVs that may affect drug binding, protein–drug complex structures were retrieved from Protein Data Bank (PDB) followed by identification of amino acids in the protein–drug binding sites using an occluded surface method. Then, the germline and somatic mutations were mapped to these amino acids to identify which of these alter protein–drug binding sites. Using this method we identified 12 993 amino acid–drug binding sites across 253 unique proteins bound to 235 unique drugs. The integration of amino acid–drug binding sites data with both germline and somatic nsSNVs data sets revealed 3133 nsSNVs affecting amino acid–drug binding sites. In addition, a comprehensive drug target discovery was conducted based on protein structure similarity and conservation of amino acid–drug binding sites. Using this method, 81 paralogs were identified that could serve as alternative drug targets. In addition, non-human mammalian proteins bound to drugs were used to identify 142 homologs in humans that can potentially bind to drugs. In the current protein–drug pairs that contain somatic mutations within their binding site, we identified 85 proteins with significant differential gene expression changes associated with specific cancer types. Information on protein–drug binding predicted drug target proteins and prevalence of both somatic and germline nsSNVs that disrupt these binding sites can provide valuable knowledge for personalized medicine treatment. A web portal is available where nsSNVs from individual patient can be checked by scanning against DrugVar to determine whether any of the SNVs affect the binding of any drug in the database.

Protein sequence conservation and stable molecular evolution reveals influenza virus nucleoprotein as a universal druggable target

The high mutation rate in influenza virus genome and appearance of drug resistance calls for a constant effort to identify alternate drug targets and develop new antiviral strategies. The internal proteins of the virus can be exploited as a potential target for therapeutic interventions. Among these, the nucleoprotein (NP) is the most abundant protein that provides structural and functional support to the viral replication machinery. The current study aims at analysis of protein sequence polymorphism patterns, degree of molecular evolution and sequence conservation as a function of potential druggability of nucleoprotein. We analyzed a universal set of amino acid sequences, (n=22,000) and, in order to identify and correlate the functionally conserved, druggable regions across different parameters, classified them on the basis of host organism, strain type and continental region of sample isolation. The results indicated that around 95% of the sequence length was conserved, with at least 7 regions conserved across the protein among various classes. Moreover, the highly variable regions, though very limited in number, were found to be positively selected indicating, thereby, the high degree of protein stability against various hosts and spatio-temporal references. Furthermore, on mapping the conserved regions on the protein, 7 drug binding pockets in the functionally important regions of the protein were revealed. The results, therefore, collectively indicate that nucleoprotein is a highly conserved and stable viral protein that can potentially be exploited for development of broadly effective antiviral strategies.

Abstract 682: Pharmacological Inhibition of COX and LOX Pathways Leads to Substrate Rediversion: A Novel Plasma Lipidomics Assay to Screen Drugs in Human Blood

Cardiovascular complications from cyclooxygenase (COX)-2-selective nonsteroidal anti-inflammatory drugs (NSAIDs) prompted interest in microsomal prostaglandin synthase 1 (mPGES-1), downstream of COX-2, as an alternative drug target. Unlike COX-2, global deletion of mPGES-1 in mice suppresses prostaglandin E2 (PGE2) and augments prostacyclin PGI2 through substrate rediversion, and does not predispose to thrombogenesis and hypertension. However, after cell-specific deletions of mPGES-1, the predominant substrate rediversion product amongst the prostaglandins varies by cell type. We hypothesized that inhibition of mPGES-1 leads to changes in bioactive lipids beyond the prostaglandin pathway, and that substrate rediversion associated with a more favorable cardiovascular profile after mPGES-1 vs COX-2 inhibition can be translated from mouse to human studies. In this project, we aimed 1) to develop a mass spectrometry-based, broad-spectrum lipidomics approach to measure arachidonate-derived products additional to prostanoids; and 2) to work out an in vitro human whole blood assay (hWBA) to study substrate rediversion upon mPGES-1 vs COX-2 inhibition. We have developed a novel, reproducible analytical method to scan the impact of drugs on the bioactive lipidome. Using an in vitro hWBA and HPLC-MS/MS lipidomics analysis, we observed substrate rediversion in both COX and LOX pathways after mPGES-1 inhibition, which was distinct from inhibiting COX-1/2. We further optimized this assay to screen combinations of inhibitors from COX and lipoxygenase (LOX) cascades. Overall, we have shown that a clear signature of inhibiting diverse targets - mPGES-1, COX and LOX enzymes - can be readily detected from experiments of small samples sizes, and discovered novel aspects of drug effect due to substrate rediversion. Our findings contribute to the comparative cardiovascular safety of inhibiting mPGES-1 vs COX-2 and demonstrate the impact of combining mPGES-1 with LOX inhibitions on the hemostatic system

EZH2 inhibition sensitizes BRG1 and EGFR mutant lung tumours to TopoII inhibitors.

SUMMARYNon-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide1. Chemotherapies such as the topoisomerase II inhibitor (TopoIIi) etoposide effectively reduce disease in a minority of NSCLC patients2,3; therefore, alternative drug targets, including epigenetic enzymes, are under consideration for therapeutic intervention4. A promising potential epigenetic target is the methyltransferase EZH2, which in the context of the Polycomb Repressive Complex 2 (PRC2) is well known to tri-methylate Histone H3 at lysine 27 (H3K27me3) and elicit gene silencing5. Here, we demonstrate that EZH2 inhibition (EZH2i) had differential effects on TopoIIi response of NSCLCs in vitro and in vivo. EGFR and BRG1 mutations were genetic biomarkers that predicted enhanced sensitivity to TopoIIi in response to EZH2i. BRG1 loss-of-function mutant tumors responded to EZH2i with increased S phase, anaphase bridging, apoptosis, and TopoIIi sensitivity. Conversely, EGFR and BRG1 wild-type tumors up-regulated BRG1 in response to EZH2i and ultimately became more resistant to TopoIIi. EGFR gain-of-function mutant tumors were also sensitive to dual EZH2i and TopoIIi, due to genetic antagonism between EGFR and BRG1. These findings suggest an exciting opportunity for precision medicine in the genetically complex disease of NSCLC.

Prediction of riboswitch as a potential drug target and design of its optimal inhibitors for Mycobacterium tuberculosis

Non-homologous proteins have been widely identified as major drug targets in host-parasite diseases like tuberculosis. The available antibiotic treatment for infectious diseases have suffered a major setback due to the development of drug resistance by the pathogen. Therefore, in the present study the riboswitch is explored as a promising alternative potential drug target. A framework has been developed for the prediction of riboswitch as a potential drug target and design of corresponding inhibitor for M. tuberculosis bacteria by molecular docking simulation based virtual screening. The virtual screening is performed by using molecular library containing 5017 diverse ligands against tuberculosis bacterial riboswitch. Thus, it is concluded that the riboswitches can be alternative potential drug-target for infectious disease to overcome the present issues of existing antibiotic drugs like their side effects and drug resistance, etc. Further a single riboswitch can be a drug target for multiple strains of bacteria and other pathogenic organisms.

SLC6A14 Modifies Fluid Secretory Capacity of Cystic Fibrosis Affected Epithelium by Enhancing CFTR Channel Function

Cystic fibrosis (CF) is usually caused by F508del mutation in the CFTR gene. Normally, CFTR protein aids in salt transport across the apical surface of epithelia, keeping their surface hydrated. F508del results in decreased surface expression of CFTR, leading to dehydration of the surface. This makes the lung epithelium prone to infection and in intestine leads to obstruction. Patients homozygous for F508del, have a tremendous variation in the severity of disease. Recent Genome-wide association studies indicate that this variation is due to presence of modifier genes, with SLC6A14 as the top modifier (Sun et.al.,2012). SLC6A14 is a Na+/Cl- dependent cationic/neutral amino-acid transporter on the surface of lung and colonic epithelium. As both transporters are expressed apically, we hypothesized that SLC6A14 would modify the fluid secretory capacity of the epithelium. So in collaboration with TCP, we generated a SLC6A14 knock-out mouse. We can measure in-vivo fluid secretion in mice, using an intestinal closed-loop assay. SLC6A14 knock-out mice exhibited a decrease in cAMP stimulated fluid secretion mediated via CFTR relative to Wt control. To explore the mechanism by which this modification occurs, we utilized a BHK heterologous expression system, overexpressing CFTR and SLC6A14. Interestingly, the functional interaction can be recapitulated in this system, suggesting that it not tissue-type dependent. Preliminary biochemical and anion-flux studies support the hypothesis that SLC6A14 does not affect the processing or stability of Wt or F508del-CFTR proteins rather it augments the activity of these channel proteins once localized to the cell surface. Future studies will focus on understanding if this augmentation is related to modification of CFTR's phosphorylation dependent gating. These results show a positive impact of SLC6A14 on CFTR channel function and fluid secretion, providing an alternative drug target for CF patients.

Positive KCa Channel Gating Modulators with Selectivity for KCa3.1

Positive KCa Channel Gating Modulators with Selectivity for KCa3.1

Global geno-proteomic analysis reveals cross-continental sequence conservation and druggable sites among influenza virus polymerases

Influenza virus is one of the major causes of mortality and morbidity associated with respiratory diseases. The high rate of mutation in the viral proteome provides it with the ability to survive in a variety of host species. This property helps it in maintaining and developing its pathogenicity, transmission and drug resistance. Alternate drug targets, particularly the internal proteins, can potentially be exploited for addressing the resistance issues. In the current analysis, the degree of conservation of influenza virus polymerases has been studied as one of the essential elements for establishing its candidature as a potential target of antiviral therapy. We analyzed more than 130,000 nucleotide and amino acid sequences by classifying them on the basis of continental presence of host organisms. Computational analyses including genetic polymorphism study, mutation pattern determination, molecular evolution and geophylogenetic analysis were performed to establish the high degree of conservation among the sequences. These studies lead to establishing the polymerases, in particular PB1, as highly conserved proteins. Moreover, we mapped the conservation percentage on the tertiary structures of proteins to identify the conserved, druggable sites. The research study, hence, revealed that the influenza virus polymerases are highly conserved (95–99%) proteins with a very slow mutation rate. Potential drug binding sites on various polymerases have also been reported. A scheme for drug target candidate development that can be employed to rapidly mutating proteins has been presented. Moreover, the research output can help in designing new therapeutic molecules against the identified targets.

Abstract 456: ERG regulation of intracrine androgen production and castration-resistant prostate cancer progression

Abstract Introduction and Objective: Intratumoral androgen synthesis in prostate cancer (PC) contributes to the development of castration-resistant prostate cancer (CRPC). Several enzymes responsible for androgen biosynthesis have been shown to be overexpressed in CRPC, thus, contributing to CRPC in a castrated environment. However, little is known regarding their mode of regulation and expression in this context. The TMPRSS2-ERG transcription factor has been shown to be present in primary PC tumors as well as CRPC tumors; however, its biological function contributing to the development CRPC in the context of intratumoral androgen synthesis is not known. We hypothesize that TMPRSS2-ERG fusions regulate androgen biosynthetic enzyme (ABE) gene expression and the production of androgens, which contributes to the development of CRPC. Methods: We used a variety of methods including lentiviral knockdown and overexpression of ERG, qPCR, Western blot, ChIP-PCR, and Mass Spectrometry. Results: We found that (a) shRNA knockdown of ERG in VCaP PC cells significantly reduces the expression of ABE's AKR1C3, HSD17B6, and HSD17B4; (b) overexpression of truncated ERG or wild-type ERG in LNCaP PC cells significantly increases the expression of AKR1C3 and HSD17B6; (c) ERG binds to the AKR1C3 gene in VCaP PC cells; and (d) DHT synthesis is reduced in VCaP shERG cells treated with 5α-Androstanedione, a DHT precursor metabolite, compared to VCaP shScrambled controls. Conclusions: These data suggest that AKR1C3 catalyzes the biochemical reduction of 5α-Androstanedione to DHT in PC cells, and that ERG may regulate this step through upregulation of AKR1C3 expression. Elucidation of ERG regulation of ABEs in CRPC may help to stratify TMPRSS2-ERG fusion-positive PC patients in the clinic for anti-hormone driven therapies. Additionally, not all PC tumors express CYP17A1 enzyme, therefore this study emphasizes the importance of investigating ABEs other than CYP17A1, such as AKR1C3 and HSD17B6. In summary, this study addresses alternative ABE drug targets as well as possible drug resistance modes in PC treatment. Citation Format: Katelyn A. Powell, Louie Semaan, Krishna R. Maddipati, Katie (Mary) Conley-Lacomb, Yanfeng Li, Michael Cher, Sreenivasa R. Chinni. ERG regulation of intracrine androgen production and castration-resistant prostate cancer progression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 456. doi:10.1158/1538-7445.AM2014-456

Cytochrome P450 monooxygenase CYP53 family in fungi: comparative structural and evolutionary analysis and its role as a common alternative anti-fungal drug target.

Cytochrome P450 monooxygenases (CYPs/P450s) are heme-thiolate proteins whose role as a drug target against pathogenic microbes has been explored because of their stereo- and regio-specific oxidation activity. We aimed to assess the CYP53 family's role as a common alternative drug target against animal (including human) and plant pathogenic fungi and its role in fungal-mediated wood degradation. Genome-wide analysis of fungal species revealed the presence of CYP53 members in ascomycetes and basidiomycetes. Basidiomycetes had a higher number of CYP53 members in their genomes than ascomycetes. Only two CYP53 subfamilies were found in ascomycetes and six subfamilies in basidiomycetes, suggesting that during the divergence of phyla ascomycetes lost CYP53 P450s. According to phylogenetic and gene-structure analysis, enrichment of CYP53 P450s in basidiomycetes occurred due to the extensive duplication of CYP53 P450s in their genomes. Numerous amino acids (103) were found to be conserved in the ascomycetes CYP53 P450s, against only seven in basidiomycetes CYP53 P450s. 3D-modelling and active-site cavity mapping data revealed that the ascomycetes CYP53 P450s have a highly conserved protein structure whereby 78% amino acids in the active-site cavity were found to be conserved. Because of this rigid nature of ascomycetes CYP53 P450s' active site cavity, any inhibitor directed against this P450 family can serve as a common anti-fungal drug target, particularly toward pathogenic ascomycetes. The dynamic nature of basidiomycetes CYP53 P450s at a gene and protein level indicates that these P450s are destined to acquire novel functions. Functional analysis of CYP53 P450s strongly supported our hypothesis that the ascomycetes CYP53 P450s ability is limited for detoxification of toxic molecules, whereas basidiomycetes CYP53 P450s play an additional role, i.e. involvement in degradation of wood and its derived components. This study is the first report on genome-wide comparative structural (gene and protein structure-level) and evolutionary analysis of a fungal P450 family.