Rational Development of Novel Activity Probes for the Analysis of Human Cytochromes P450

The identification and quantification of functional cytochromes P450 (CYPs) in biological samples is proving important for robust analyses of drug efficacy and metabolic disposition. In this study, a novel CYP activity‐based probe was rationally designed and synthesised, demonstrating selective binding of CYP isoforms. The dependence of probe binding upon the presence of NADPH permits the selective detection of functionally active CYP. This allows the detection and analysis of these enzymes using biochemical and proteomic methodologies and approaches.     

Two‐Step Labeling of Endogenous Enzymatic Activities by Diels–Alder Ligation

A ligation strategy based on the Diels–Alder [4+2] cycloaddition for the two‐step activity‐based labeling of endogenously expressed enzymes in complex biological samples has been developed. A panel of four diene‐derivatized proteasome probes was synthesized, along with a dienophile‐functionalized BODIPY(TMR) tag. These probes were applied in a Diels–Alder labeling procedure that enabled us to label active proteasome β‐subunits selectively in cellular extracts and in living cells. We were also able to label the activity of cysteine proteases in cell extracts by utilizing a diene‐derivatized cathepsin probe. Importantly, the Diels–Alder strategy described here is fully orthogonal with respect to the Staudinger–Bertozzi ligation, as demonstrated by the independent labeling of different proteolytic activities by the two methods in a single experiment.     

Inhibition of Mycobacterium tuberculosis methionine aminopeptidases by bengamide derivatives

Methionine aminopeptidase (MetAP) carries out an essential function of protein N‐terminal processing in many bacteria and is a promising target for the development of novel antitubercular agents. Natural bengamides potently inhibit the proliferation of mammalian cells by targeting MetAP enzymes, and the X‐ray crystal structure of human type 2 MetAP in complex with a bengamide derivative reveals the key interactions at the active site. By preserving the interactions with the conserved residues inside the binding pocket while exploring the differences between bacterial and human MetAPs around the binding pocket, seven bengamide derivatives were synthesized and evaluated for inhibition of MtMetAP1a and MtMetAP1c in different metalloforms, inhibition of M. tuberculosis growth in replicating and non‐replicating states, and inhibition of human K562 cell growth. Potent inhibition of MtMetAP1a and MtMetAP1c and modest growth inhibition of M. tuberculosis were observed for some of these derivatives. Crystal structures of MtMetAP1c in complex with two of the derivatives provided valuable structural information for improvement of these inhibitors for potency and selectivity.     

Mass Spectrometry-Based Proteomics for Pre-Eclampsia and Preterm Birth

Pregnancy-related complications such as pre-eclampsia and preterm birth now represent a notable burden of adverse health. Pre-eclampsia is a hypertensive disorder unique to pregnancy. It is an important cause of maternal death worldwide and a leading cause of fetal growth restriction and iatrogenic prematurity. Fifteen million infants are born preterm each year globally, but more than one million of those do not survive their first month of life. Currently there are no predictive tests available for diagnosis of these pregnancy-related complications and the biological mechanisms of the diseases have not been fully elucidated. Mass spectrometry-based proteomics have all the necessary attributes to provide the needed breakthrough in understanding the pathophysiology of complex human diseases thorough the discovery of biomarkers. The mass spectrometry methodologies employed in the studies for pregnancy-related complications are evaluated in this article. Top-down proteomic and peptidomic profiling by laser mass spectrometry, liquid chromatography or capillary electrophoresis coupled to mass spectrometry, and bottom-up quantitative proteomics and targeted proteomics by liquid chromatography mass spectrometry have been applied to elucidate protein biomarkers and biological mechanism of pregnancy-related complications. The proteomes of serum, urine, amniotic fluid, cervical-vaginal fluid, placental tissue, and cytotrophoblastic cells have all been investigated. Numerous biomarkers or biomarker candidates that could distinguish complicated pregnancies from healthy controls have been proposed. Nevertheless, questions as to the clinically utility and the capacity to elucidate the pathogenesis of the pre-eclampsia and preterm birth remain to be answered.