ABSTRACT
Experiments comprising a "pre-incubation" phase, where enzyme is incubated with inhibitor prior to the addition of assay substrate, are commonly used to evaluate covalent inhibitors, often via discontinuous or "endpoint" IC50 assays. However, due to the lack of mathematical tools to describe its biphasic time-dependent nature, this experiment has thus far been unable to provide kinact and KI values. Herein we report EPIC-Fit, a new method to determine kinact and KI values from global fitting of Endpoint Pre-incubation IC50 data that can be implemented using Microsoft Excel. Experimental characterization of a known tissue transglutaminase inhibitor, AA9, using EPIC-Fit provided kinact and KI values with strong correlations to the values determined by other, previously established methods of evaluation. This unprecedented method serves to finally include time-dependent pre-incubation endpoint assays in the medicinal chemist's toolbox for rigorous characterization of irreversible inhibitors.
ABSTRACT
Human tissue transglutaminase (hTG2) is a multifunctional enzyme with protein cross-linking and G-protein activity, both of which have been implicated in the progression of diseases such as fibrosis and cancer stem cell propagation when dysregulated, prompting the development of small molecule targeted covalent inhibitors (TCIs) possessing a crucial electrophilic 'warhead'. In recent years there have been significant advances in the library of warheads available for the design of TCIs; however, the exploration of warhead functionality in hTG2 inhibitors has remained relatively stagnant. Herein, we describe a structure-activity relationship study entailing rational design and synthesis for systematic variation of the warhead on a previously reported small molecule inhibitor scaffold, and rigorous kinetic evaluation of inhibitory efficiency, selectivity, and pharmacokinetic stability. This study reveals a strong influence on the kinetic parameters k inact and K I with even subtle variation in warhead structure, suggesting that the warhead plays a significant role in not only reactivity, but also binding affinity, which consequently extends to isozyme selectivity. Warhead structure also influences in vivo stability, which we model by measuring intrinsic reactivity with glutathione, as well as stability in hepatocytes and in whole blood, giving insight into degradation pathways and relative therapeutic potential of different functional groups. This work provides fundamental structural and reactivity information highlighting the importance of strategic warhead design for the development of potent hTG2 inhibitors.
ABSTRACT
Prostate-specific membrane antigen (PSMA) is highly overexpressed in most prostate cancers and is clinically visualized using PSMA-specific probes incorporating glutamate-ureido-lysine (GUL). PSMA is effectively absent from certain high-mortality, treatment-resistant subsets of prostate cancers, such as neuroendocrine prostate cancer (NEPC); however, GUL-based PSMA tracers are still reported to have the potential to identify NEPC metastatic tumors. These probes may bind unknown proteins associated with PSMA-suppressed cancers. We have identified the up-regulation of PSMA-like aminopeptidase NAALADaseL and the metabotropic glutamate receptors (mGluRs) in PSMA-suppressed prostate cancers and find that their expression levels inversely correlate with PSMA expression and are associated with GUL-based radiotracer uptake. Furthermore, we identify that NAALADaseL and mGluR expression correlates with a unique cell cycle signature. This provides an opportunity for the future study of the biology of NEPC and potential therapeutic directions. Computationally predicting that GUL-based probes bind well to these targets, we designed and synthesized a fluorescent PSMA tracer to investigate these proteins in vitro, where it shows excellent affinity for PSMA, NAALADaseL, and specific mGluRs associated with poor prognosis.
