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.
BackgroundBacteriophages (phages) have been used extensively as analytical tools to type bacterial cultures and recently for control of zoonotic foodborne pathogens in foods and in animal reservoirs.MethodsWe examined the host range, morphology, genome and proteome of the lytic E. coli O157 phage rV5, derived from phage V5, which is a member of an Escherichia coli O157:H7 phage typing set.ResultsPhage rV5 is a member of the Myoviridae family possessing an icosahedral head of 91 nm between opposite apices. The extended tail measures 121 x 17 nm and has a sheath of 44 x 20 nm and a 7 nm-wide core in the contracted state. It possesses a 137,947 bp genome (43.6 mol%GC) which encodes 233 ORFs and six tRNAs. Until recently this virus appeared to be phylogenetically isolated with almost 70% of its gene products ORFans. rV5 is closely related to coliphages Delta and vB-EcoM-FY3, and more distantly related to Salmonella phages PVP-SE1 and SSE-121, Cronobacter sakazakii phage vB_CsaM_GAP31, and coliphages phAPEC8 and phi92. A complete shotgun proteomic analysis was carried out on rV5, extending what had been gleaned from the genomic analyses. Host range studies revealed that rV5 is active against several other E. coli.
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.
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