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1.
ACS Chem Biol ; 17(9): 2595-2604, 2022 09 16.
Article in English | MEDLINE | ID: mdl-36044633

ABSTRACT

Although current antiretroviral therapy can control HIV-1 replication and prevent disease progression, it is not curative. Identifying mechanisms that can lead to eradication of persistent viral reservoirs in people living with HIV-1 (PLWH) remains an outstanding challenge to achieving cure. Utilizing a phenotypic screen, we identified a novel chemical class capable of killing HIV-1 infected peripheral blood mononuclear cells. Tool compounds ICeD-1 and ICeD-2 ("inducer of cell death-1 and 2"), optimized for potency and selectivity from screening hits, were used to deconvolute the mechanism of action using a combination of chemoproteomic, biochemical, pharmacological, and genetic approaches. We determined that these compounds function by modulating dipeptidyl peptidase 9 (DPP9) and activating the caspase recruitment domain family member 8 (CARD8) inflammasome. Efficacy of ICeD-1 and ICeD-2 was dependent on HIV-1 protease activity and synergistic with efavirenz, which promotes premature activation of HIV-1 protease at high concentrations in infected cells. This in vitro synergy lowers the efficacious cell kill concentration of efavirenz to a clinically relevant dose at concentrations of ICeD-1 or ICeD-2 that do not result in complete DPP9 inhibition. These results suggest engagement of the pyroptotic pathway as a potential approach to eliminate HIV-1 infected cells.


Subject(s)
HIV Infections , HIV-1 , Alkynes , Benzoxazines , CARD Signaling Adaptor Proteins/metabolism , Cyclopropanes , Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/metabolism , HIV Infections/drug therapy , HIV-1/metabolism , Humans , Inflammasomes/metabolism , Leukocytes, Mononuclear , Neoplasm Proteins/metabolism
2.
J Biomol Screen ; 20(2): 212-22, 2015 Feb.
Article in English | MEDLINE | ID: mdl-25336354

ABSTRACT

As a label-free technology, mass spectrometry (MS) enables assays to be generated that monitor the conversion of substrates with native sequences to products without the requirement for substrate modifications or indirect detection methods. Although traditional liquid chromatography (LC)-MS methods are relatively slow for a high-throughput screening (HTS) paradigm, with cycle times typically ≥ 60 s per sample, the Agilent RapidFire High-Throughput Mass Spectrometry (HTMS) System, with a cycle time of 5-7 s per sample, enables rapid analysis of compound numbers compatible with HTS. By monitoring changes in mass directly, HTMS assays can be used as a triaging tool by eliminating large numbers of false positives resulting from fluorescent compound interference or from compounds interacting with hydrophobic fluorescent dyes appended to substrates. Herein, HTMS assays were developed for multiple protease programs, including cysteine, serine, and aspartyl proteases, and applied as a confirmatory assay. The confirmation rate for each protease assay averaged <30%, independent of the primary assay technology used (i.e., luminescent, fluorescent, and time-resolved fluorescent technologies). Importantly, >99% of compounds designed to inhibit the enzymes were confirmed by the corresponding HTMS assay. Hence, HTMS is an effective tool for removing detection-based false positives from ultrahigh-throughput screening, resulting in hit lists enriched in true actives for downstream dose response titrations and hit-to-lead efforts.


Subject(s)
High-Throughput Screening Assays/methods , Mass Spectrometry/methods , Peptide Hydrolases/metabolism , Dose-Response Relationship, Drug , Drug Discovery , Enzyme Assays/methods , Enzyme Assays/standards , High-Throughput Screening Assays/standards , Humans , Mass Spectrometry/standards , Protease Inhibitors/pharmacology , Reproducibility of Results , Substrate Specificity
3.
J Med Chem ; 55(7): 2945-59, 2012 Apr 12.
Article in English | MEDLINE | ID: mdl-22364528

ABSTRACT

The discovery of 1,3,8-triazaspiro[4.5]decane-2,4-diones (spirohydantoins) as a structural class of pan-inhibitors of the prolyl hydroxylase (PHD) family of enzymes for the treatment of anemia is described. The initial hit class, spirooxindoles, was identified through affinity selection mass spectrometry (AS-MS) and optimized for PHD2 inhibition and optimal PK/PD profile (short-acting PHDi inhibitors). 1,3,8-Triazaspiro[4.5]decane-2,4-diones (spirohydantoins) were optimized as an advanced lead class derived from the original spiroindole hit. A new set of general conditions for C-N coupling, developed using a high-throughput experimentation (HTE) technique, enabled a full SAR analysis of the spirohydantoins. This rapid and directed SAR exploration has resulted in the first reported examples of hydantoin derivatives with good PK in preclinical species. Potassium channel off-target activity (hERG) was successfully eliminated through the systematic introduction of acidic functionality to the molecular structure. Undesired upregulation of alanine aminotransferese (ALT) liver enzymes was mitigated and a robust on-/off-target margin was achieved. Spirohydantoins represent a class of highly efficacious, short-acting PHD1-3 inhibitors causing a robust erythropoietin (EPO) upregulation in vivo in multiple preclinical species. This profile deems spirohydantoins as attractive short-acting PHDi inhibitors with the potential for treatment of anemia.


Subject(s)
Anemia/drug therapy , Aza Compounds/chemical synthesis , Hydantoins/chemical synthesis , Hypoxia-Inducible Factor 1/metabolism , Procollagen-Proline Dioxygenase/antagonists & inhibitors , Spiro Compounds/chemical synthesis , Animals , Aza Compounds/pharmacokinetics , Aza Compounds/pharmacology , Dogs , ERG1 Potassium Channel , Erythropoietin/biosynthesis , Ether-A-Go-Go Potassium Channels/metabolism , High-Throughput Screening Assays , Humans , Hydantoins/pharmacokinetics , Hydantoins/pharmacology , Hypoxia-Inducible Factor-Proline Dioxygenases , Indoles/chemical synthesis , Indoles/pharmacokinetics , Indoles/pharmacology , Liver/drug effects , Liver/enzymology , Macaca mulatta , Mass Spectrometry , Mice , Mice, Inbred C57BL , Protein Binding , Rats , Spiro Compounds/pharmacokinetics , Spiro Compounds/pharmacology , Structure-Activity Relationship , Up-Regulation
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