SLoMo: Automated Site Localization of Modifications from ETD/ECD Mass Spectra

Recently, software has become available to automate localization of phosphorylation sites from CID data and to assign associated confidence scores. We present an algorithm, SLoMo (Site Localization of Modifications), which extends this capability to ETD/ECD mass spectra. Furthermore, SLoMo caters for both high and low resolution data and allows for site-localization of any UniMod post-translational modification. SLoMo accepts input data from a variety of formats (e.g., Sequest, OMSSA). We validate SLoMo with high and low resolution ETD, ECD, and CID data

SLoMo: Automated Site Localization of Modifications from ETD/ECD Mass Spectra

Recently, software has become available to automate localization of phosphorylation sites from CID data and to assign associated confidence scores. We present an algorithm, SLoMo (Site Localization of Modifications), which extends this capability to ETD/ECD mass spectra. Furthermore, SLoMo caters for both high and low resolution data and allows for site-localization of any UniMod post-translational modification. SLoMo accepts input data from a variety of formats (e.g., Sequest, OMSSA). We validate SLoMo with high and low resolution ETD, ECD, and CID data

Modulation of Human Immunodeficiency Virus Type 1 Synergistic Inhibition by Reverse Transcriptase Mutations^†

Synergy between the anti-human immunodeficiency virus type 1 (HIV) nucleoside reverse transcriptase (RT) inhibitors (NRTIs) and nonnucleoside RT inhibitors (NNRTIs) results from a general mechanism in which NNRTIs inhibit ATP-mediated removal of NRTIs from chain-terminated primers by decreasing the maximum rate of removal, thus sustaining NRTI chain termination. With this molecular mechanism of synergy, β-d-(+)-3‘-azido-3‘-deoxythymidine monophosphate (AZTMP) removal was examined in the context of clinically relevant RT mutants. The IC50 value for inhibition by nevirapine against wild-type (WT) RT in our removal assay was 3 μM, but this concentration had no effect on removal by the nevirapine-resistant Y181C mutant. Rather, a ∼83-fold increase in nevirapine was required to decrease the rate of removal by 50% for this mutant. Efavirenz displayed a 100 nM IC50 value against WT and the efavirenz-sensitive Y181C mutant, but the efavirenz-resistant mutants K103N and K103N/Y181C required a 6-fold increase in efavirenz concentration to achieve the same effect. A newer generation NNRTI, TMC125, showed potency (55 nM) against WT and all mutants, paralleling the activity of this inhibitor relative to nevirapine and efavirenz in cell culture. When tested against the AZT-resistant mutant, all NNRTIs inhibited removal by greater than 50%, showing that this mutant is hypersensitive to NNRTIs. Altogether these results illustrate that both the NNRTI and NRTI mutations can modulate chain termination. This demonstrates that sustaining synergistic HIV inhibition in combination NRTI/NNRTI therapy requires NNRTIs that are potent against WT virus and possess favorable activity profiles against clinically relevant mutations

FEP-Guided Selection of Bicyclic Heterocycles in Lead Optimization for Non-Nucleoside Inhibitors of HIV-1 Reverse Transcriptase

Monte Carlo simulations using free energy perturbation theory have been used to guide the selection of bicyclic heterocycles in the lead optimization of non-nucleoside inhibitors of HIV-1 reverse transcriptase (NNRTIs). Good correlation is found between predicted and observed activities. Six compounds are reported with EC50 values below 20 nM for protection of human MT-2 cells against the cytopathogenicity of HIV-1. Striking variation in activity is found and analyzed for an isomeric pyrrolopyrimidine and pyrrolopyrazine pair

From Docking False-Positive to Active Anti-HIV Agent

Virtual screening of the Maybridge library of ca. 70 000 compounds was performed using a similarity filter, docking, and molecular mechanics−generalized Born/surface area postprocessing to seek potential non-nucleoside inhibitors of human immunodeficiency virus-1 (HIV-1) reverse transcriptase (NNRTIs). Although known NNRTIs were retrieved well, purchase and assaying of representative, top-scoring compounds from the library failed to yield any active anti-HIV agents. However, the highest-ranked library compound, oxadiazole 1, was pursued as a potential “near-miss” with the BOMB program to seek constructive modifications. Subsequent synthesis and assaying of several polychloro-analogs did yield anti-HIV agents with EC50 values as low as 310 nM. The study demonstrates that it is possible to learn from a formally unsuccessful virtual-screening exercise and, with the aid of computational analyses, to efficiently evolve a false positive into a true active

Optimization of Azoles as Anti-Human Immunodeficiency Virus Agents Guided by Free-Energy Calculations

Efficient optimization of an inactive 2-anilinyl-5-benzyloxadiazole core has been guided by free energy perturbation (FEP) calculations to provide potent non-nucleoside inhibitors of human immunodeficiency virus (HIV) reverse transcriptase (NNRTIs). An FEP “chlorine scan” was performed to identify the most promising sites for substitution of aryl hydrogens. This yielded NNRTIs 8 and 10 with activities (EC50) of 820 and 310 nM for protection of human T-cells from infection by wild-type HIV-1. FEP calculations for additional substituent modifications and change of the core heterocycle readily led to oxazoles 28 and 29, which were confirmed as highly potent anti-HIV agents with activities in the 10−20 nM range. The designed compounds were also monitored for possession of desirable pharmacological properties by use of additional computational tools. Overall, the trends predicted by the FEP calculations were well borne out by the assay results. FEP-guided lead optimization is confirmed as a valuable tool for molecular design including drug discovery; chlorine scans are particularly attractive since they are both straightforward to perform and highly informative

Supplementary Tables and Figures from PP2A-activating Drugs Enhance FLT3 Inhibitor Efficacy through AKT Inhibition–Dependent GSK-3β–Mediated c-Myc and Pim-1 Proteasomal Degradation

Supplementary Table S1. AML patients. Supplementary Table S2. RT-qPCR primers. Supplementary Table S3. IC50 concentrations (nM) of FLT3 inhibitors, PP2A activators and GSK-3beta inhibitors. Supplementary Figure S1. Concurrent PP2A-activating drug treatment increases cytotoxicity of FLT3 inhibitors in Ba/F3-ITD cells with FLT3-ITD and in an MV4-11 orthotopic mouse model. Supplementary Figure S2. Chou-Talalay analysis of FLT3-WT cell lines. Supplementary Figure S3. Images of all mice in the in vivo experiment. Supplementary Figure S4. Concurrent treatment with PP2A-activating drug and FLT3 inhibitor does not induce apoptosis in wild-type FLT3 AML, AML complete remission (CR) or AML complete remission with incomplete platelet recovery (CRp) marrows. Supplementary Figure S5. Concurrent treatment with PP2A-activating drug and FLT3 inhibitor downregulates c-Myc and Pim-1 protein expression in cells with FLT3-ITD. Supplementary Figure S6. p-c-Myc (T58) and p-c-Myc (S62) expression in Ba/F3-ITD and MV4-11 cells treated with gilteritinib and/or FTY720. Supplementary Figure S7. Concurrent treatment with PP2A-activating drug and FLT3 inhibitor produces variable changes in c-Myc and Pim-1 mRNA expression in cells with FLT3-ITD. Supplementary Figure S8. c-Myc and Pim-1 expression in FLT3-WT AML blasts treated with gilteritinib and/or FTY720. Supplementary Figure S9. Proteasome inhibition inhibits downregulation of c-Myc and Pim-1 expression by concurrent PP2A-activating drug and FLT3 inhibitor treatment. Supplementary Figure S10. Concurrent PP2A-activating drug and FLT3 inhibitor treatment increases c-Myc and Pim-1 protein turnover in cells with FLT3-ITD. Supplementary Figure S11. Concurrent PP2A activator and FLT3 inhibitor treatment downregulates p-ERK later than p-AKT in cells with FLT3-ITD. Supplementary Figure S12. The GSK-3beta inhibitor TWS119 prevents c-Myc and Pim-1 downregulation and apoptosis induction by PP2A-activating drug and FLT3 inhibitor combination in Ba/F3-ITD cells.</p