Small molecule inhibitors of ATP7A as novel theraphies for cancer

Recent studies suggest that trace element copper (Cu) plays a key role in cancer progression. Cu dependent signaling pathways in cancer cells are poorly understood and are being actively investigated. In a recently published PNAS paper, we demonstrated that ATP7A copper transporter promotes breast and lung tumor growth and spread in mice by activating pro cancerous enzyme, lysyl oxidase (LOX). Therefore, we propose that blocking ATP7A using small molecule inhibitors could be a powerful approach in blocking cancer progression. We screened for inhibitors of ATP7A using a structure based virtual screen of ATP7A interacting drug like compounds, resulting in identification of our lead compound, MKV3. We used microscale thermophoresis and standard enzyme activity assays to measure the binding affinity of MKV3 to ATP7A and test the effect of MKV3 on ATP7A dependent LOX and tyrosinase enzymes. In vitro scratch and tumorigenesis assays were performed to test the effect of MKV3 on cancer cell migration and tumor growth. Microscale thermophoresis experiments revealed that MKV3 binds to ATP7A with nanomolar affinity and is a potent inhibitor of ATP7A dependent LOX activity and cell migration of 4T1 breast, LLC and A549 lung cancer cells. Moreover, MKV3 inhibited ATP7A dependent tyrosinase activity in B16 melanoma cells and suppressed B16 tumorigenesis in vivo. In summary, these studies have identified a novel first in class high affinity inhibitor of ATP7A and provide a framework to design MKV3 derivative s with improved therapeutic efficacy in mouse models of cancer. Our findings have the potential for a sustained and powerful impact in cancer therapy.NIH R 01 fundingShanbhag Vinit, 1,4 ; Gudekar Nikita 2 ; Kamlendra Singh 3 ; Petris, Michael 1,2,4 (1 - Department of Biochemistry, 2 - Genetics Area Program, 3 - Department of Veterinary Pathobiology, 4 - Department of Opthalmology, University of Missouri, Columbia 65211

Drug resistance mutations against protease, reverse transcriptase and integrase inhibitors in people living with HIV-1 receiving boosted protease inhibitors in South Africa

The South African national combination antiretroviral therapy (cART) roll-out program started in 2006, with over 4.4 million people accessing treatment since it was first introduced. HIV-1 drug resistance can hamper the success of cART. This study determined the patterns of HIV-1 drug-resistance associated mutations (RAMs) in People Living with HIV-1 (PLHIV-1). Receiving first (for children below 3 years of age) and second-line (for adults) cART regimens in South Africa. During 2017 and 2018, 110 patients plasma samples were selected, 96 samples including those of 17 children and infants were successfully analyzed. All patients were receiving a boosted protease inhibitor (bPI) as part of their cART regimen. The viral sequences were analyzed for RAMs through genotypic resistance testing. We performed genotypic resistance testing (GRT) for Protease inhibitors (PIs), Reverse transcriptase inhibitors (RTIs) and Integrase strand transfer inhibitors (InSTIs). Viral sequences were subtyped using REGAv3 and COMET. Based on the PR/RT sequences, HIV-1 subtypes were classified as 95 (99%) HIV-1 subtype C (HIV-1C) while one sample as 02_AG

Discovery and Mechanistic Characterization of Novel SARS Coronavirus Inhibitors that Block Viral Entry [abstract]

Comparative Medicine - OneHealth and Comparative Medicine Poster SessionSevere acute respiratory syndrome (SARS) is an infectious and highly contagious disease that is caused by SARS-associated coronavirus (SARS-CoV). Viral entry is a key target step for therapies because it can prevent the propagation of virus at early stages of the disease. We used a cell-based assay to identify inhibitors of SARS-CoV entry. We prepared a pseudotyped virus in which the core is from HIV and envelop is from the SARS-CoV (HIV-luc/SARS env). This pseudotyped virus was used to infect, 293T cells expressing the receptor for SARS-CoV, Aangiotensin-converting enzyme-2 (ACE2). Using this assay we screened a chemical library of more than 2000 compounds and identified three compounds that specifically inhibit entry of the HIV-luc/SARS env. These compounds did not inhibit another pseudotyped virus which had same core from HIV but envelop was from Vesicular Stomatitis Virus. The compounds had strong potencies (EC50s were 2.9, 4.8 and 5.8 µM) and low cytotoxicities (high CC50s) resulting in promising Selectivity Indices (CC50/EC50 were >175, >65, and >86, respectively). Importantly, the compounds were found to have excellent antiviral activities, blocking SARS-CoV replication at low nM concentrations. Only one of the compounds was a moderate inhibitor of cathepsin L, a cellular protease whose activity is required to process the SARS-CoV env glycoprotein (Spike) and allow viral entry. Moreover, none of the compounds affects the cleavage activity of furin, another host protease, which may also be involved in SARS-CoV entry. Using a flow cytometry binding assay, we found that all three compounds decrease binding of the SARS-CoV Spike receptor binding domain to ACE2 receptor expressed on the surface of 293Tcells. Hence, we have discovered three promising compounds as the first small molecule inhibitors that can block receptor-dependent entry of SARS-CoV

A Bioinformatics Resource for TWEAK-Fn14 Signaling Pathway

TNF-related weak inducer of apoptosis (TWEAK) is a new member of the TNF superfamily. It signals through TNFRSF12A, commonly known as Fn14. The TWEAK-Fn14 interaction regulates cellular activities including proliferation, migration, differentiation, apoptosis, angiogenesis, tissue remodeling and inflammation. Although TWEAK has been reported to be associated with autoimmune diseases, cancers, stroke, and kidney-related disorders, the downstream molecular events of TWEAK-Fn14 signaling are yet not available in any signaling pathway repository. In this paper, we manually compiled from the literature, in particular those reported in human systems, the downstream reactions stimulated by TWEAK-Fn14 interactions. Our manual amassment of the TWEAK-Fn14 pathway has resulted in cataloging of 46 proteins involved in various biochemical reactions and TWEAK-Fn14 induced expression of 28 genes. We have enabled the availability of data in various standard exchange formats from NetPath, a repository for signaling pathways. We believe that this composite molecular interaction pathway will enable identification of new signaling components in TWEAK signaling pathway. This in turn may lead to the identification of potential therapeutic targets in TWEAK-associated disorders

Novel inhibitors of Foot and Mouth Disease Virus (FMDV) Targeting the RNA-Dependent RNA Polymerase activity of 3Dpol [abstract]

Comparative Medicine - OneHealth and Comparative Medicine Poster SessionFoot-and-Mouth Disease Virus (FMDV) is a positive stranded picornavirus which infects cloven-hoofed animals, such as cattle, pigs and sheep, and leads to severe losses in livestock production. In the case of an FMD outbreak, emergency vaccination could be used but it would require at least 7 days to trigger an effective immune response. On the contrary, the use of antiviral drugs is expected to have prophylactic and/or therapeutic effects almost immediately. However, there are currently no approved FMDV inhibitors. Here we have applied a combination of screening, biochemical, virological, and molecular modeling tools to discover, validate, and characterize novel inhibitors of FMDV replication. Using a luciferase-based assay we have screened a chemical library of compounds and have identified two compounds, 5-chloro-3-(thiophen-2-yl-sulfanylmethyl)-1-benzothiophene 1,1-dioxide (or C7F8) and N'1-thieno[2,3-d]pyrimidin-4-yl-4-chloro-1-benzenesulfonohydrazide (or C5D9) that inhibited the RNA-dependent RNA polymerase activity of FMDV replicase (3Dpol) with IC50 values of 2.5 μM and 15 μM respectively. These compounds were shown to be specific inhibitors of FMDV 3Dpol and not nucleic acid chelators, as they did not affect activity of other viral polymerases using the same nucleic acid substrate. Molecular modeling docking experiments suggest that both inhibitors bind at a pocket proximal to, but distinct from, the NTP binding site of 3Dpol, thereby affecting indirectly RNA synthesis. C7F8 and C5d9 were not cytotoxic at concentrations up to at least 100 ||M. Importantly, C5D9 exhibited antiviral activity and suppressed virus production in FMDV-infected cells with 50% and 90% effective concentrations (EC50 and EC90) of 10 ||M and 20 ||M, respectively. The results indicate that 3Dpol inhibitors can be promising anti-FMDV agents for use as alternative or supplementary options to contain future outbreaks of FMD

Physiological Mg²⁺ Conditions Significantly Alter the Inhibition of HIV‑1 and HIV‑2 Reverse Transcriptases by Nucleoside and Non-Nucleoside Inhibitors <i>in Vitro</i>

Reverse transcriptases (RTs) are typically assayed <i>in vitro</i> with 5–10 mM Mg²⁺, whereas the free Mg²⁺ concentration in cells is much lower. Artificially high Mg²⁺ concentrations used <i>in vitro</i> can misrepresent different properties of human immunodeficiency virus (HIV) RT, including fidelity, catalysis, pausing, and RNase H activity. Here, we analyzed nucleoside (NRTIs) and non-nucleoside RT inhibitors (NNRTIs) in primer extension assays at different concentrations of free Mg²⁺. At low concentrations of Mg²⁺, NRTIs and dideoxynucleotides (AZTTP, ddCTP, ddGTP, and 3TCTP) inhibited HIV-1 and HIV-2 RT synthesis less efficiently than they did with large amounts of Mg²⁺, whereas inhibition by the “translocation-defective RT inhibitor” EFdA (4′-ethynyl-2-fluoro-2′-deoxyadenosine) was unaffected by Mg²⁺ concentrations. Steady-state kinetic analyses revealed that the reduced level of inhibition at low Mg²⁺ concentrations resulted from a 3–9-fold (depending on the particular nucleotide and inhibitor) less efficient incorporation (based on <i>k</i>_cat/<i>K</i>_m) of these NRTIs under this condition compared to incorporation of natural dNTPs. In contrast, EFdATP was incorporated with an efficiency similar to that of its analogue dATP at low Mg²⁺ concentrations. Unlike NRTIs, NNRTIs (nevirapine, efavirenz, and rilviripine), were approximately 4-fold (based on IC₅₀ values) more effective at low than at high Mg²⁺ concentrations. Drug-resistant HIV-1 RT mutants also displayed the Mg²⁺-dependent difference in susceptibility to NRTIs and NNRTIs. In summary, analyzing the efficiency of inhibitors under more physiologically relevant low-Mg²⁺ conditions yielded results dramatically different from those from measurements using commonly employed high-Mg²⁺ <i>in vitro</i> conditions. These results also emphasize differences in Mg²⁺ sensitivity between the translocation inhibitor EFdATP and other NRTIs

Exploring 2D and 3D QSARs of benzimidazole derivatives as transient receptor potential melastatin 8 (TRPM8) antagonists using MLR and kNN-MFA methodology

TRPM8 is now best known as a cold- and menthol-activated channel implicated in thermosensation. TRPM8 is specifically expressed in a subset of pain- and temperature-sensing neuron. TRPM8 plays a major role in the sensation of cold and cooling substances. TRPM8 is a potential new target for the treatment of painful conditions. Thus, TRPM8 antagonists represent a new, novel and potentially useful treatment strategy to treat various disease states such as urological disorders, asthma, COPD, prostate and colon cancers, and painful conditions related to cold, such as cold allodynia and cold hyperalgesia. Better tools such as potent and specific TRPM8 antagonists are mandatory as high unmet medical need for such progress. To achieve this objective quantitative structure–activity relationship (QSAR) studies were carried out on a series of 25 benzimidazole-containing TRPM8 antagonists to investigate the structural requirements of their inhibitory activity against cTRPM8. The statistically significant best 2D-QSAR model having correlation coefficient r2 = 0.88 and cross-validated squared correlation coefficient q2 = 0.64 with external predictive ability of pred_r2 = 0.69 was developed by SW-MLR. The physico-chemical descriptors such as polarizabilityAHP, kappa2, XcompDipole, +vePotentialSurfaceArea, XKMostHydrophilic were found to show a significant correlation with biological activity in benzimidazole derivatives. Molecular field analysis was used to construct the best 3D-QSAR model using SW-kNN method, showing good correlative and predictive capabilities in terms of q2 = 0.81 and pred_r2 = 0.55. Developed kNN-MFA model highlighted the importance of shape of the molecules, i.e., steric & electrostatic descriptors at the grid points S_774 & E_1024 for TRPM8 receptor binding. These models (2D & 3D) were found to yield reliable clues for further optimization of benzimidazole derivatives in the data set. The information rendered by 2D- and 3D-QSAR models may lead to a better understanding of structural requirements of cTRPM8 antagonists and also can help in the design of novel potent cTRPM8 antagonists

Divergent Evolution in Reverse Transcriptase (RT) of HIV-1 Group O and M Lineages: Impact on Structure, Fitness, and Sensitivity to Nonnucleoside RT Inhibitors▿

Natural evolution in primate lentiviral reverse transcriptase (RT) appears to have been constrained by the necessity to maintain function within an asymmetric protein composed of two identical primary amino acid sequences (66 kDa), of which one is cleaved (51 kDa). In this study, a detailed phylogenetic analysis now segregates groups O and M into clusters based on a cysteine or tyrosine residue located at position 181 of RT and linked to other signature residues. Divergent evolution of two group O (C181 or Y181) and the main (Y181 only) HIV-1 lineages did not appreciably impact RT activity or function. Group O RT structural models, based on group M subtype B RT crystal structures, revealed that most evolutionarily linked amino acids appear on a surface-exposed region of one subunit while in a noncatalytic RT pocket of the other subunit. This pocket binds nonnucleoside RT inhibitors (NNRTI); therefore, NNRTI sensitivity was used to probe enzyme differences in these group O and M lineages. In contrast to observations showing acquired drug resistance associated with fitness loss, the C181Y mutation in the C181 group O lineage resulted in a loss of intrinsic NNRTI resistance and was accompanied by fitness loss. Other mutations linked to the NNRTI-resistant C181 lineage also resulted in altered NNRTI sensitivity and a net fitness cost. Based on RT asymmetry and conservation of the intricate reverse transcription process, millions of years of divergent primate lentivirus evolution may be constrained to discrete mutations that appear primarily in the nonfunctional, solvent-accessible NNRTI binding pocket