Statistical Coupling Analysis Predicts Correlated Motions in Dihydrofolate Reductase.

The role of dynamics in enzymatic function is a highly debated topic. Dihydrofolate reductase (DHFR), due to its universality and the depth with which it has been studied, is a model system in this debate. Myriad previous works have identified networks of residues in positions near to and remote from the active site that are involved in dynamics and others that are important for catalysis. For example, specific mutations on the Met20 loop in E. coli DHFR (N23PP/S148A) are known to disrupt millisecond-timescale motions and reduce catalytic activity. However, how and if networks of dynamically coupled residues influence the evolution of DHFR is still an unanswered question. In this study, we first identify, by statistical coupling analysis and molecular dynamic simulations, a network of coevolving residues, which possess increased correlated motions. We then go on to show that allosteric communication in this network is selectively knocked down in N23PP/S148A mutant E. coli DHFR. Finally, we identify two sites in the human DHFR sector which may accommodate the Met20 loop double proline mutation while preserving dynamics. These findings strongly implicate protein dynamics as a driving force for evolution.

Identification and initial characterization of a potent inhibitor of ferroptosis.

Ferroptosis is a form of iron-dependent cell death characterized by elevated lipid peroxides and reactive oxygen species (ROS). Glutathione (GSH) plays an essential role in scavenging ROS to maintain cell viability and acts as a cofactor of GSH peroxidase 4 (GPX4) that protects lipids from oxidation. We have previously described a novel class of small molecules that induce ferroptosis in certain types of cancer cells. These compounds induce ferroptosis by blocking the uptake of cystine required for GSH synthesis. Even though ferroptosis is a well-established form of cell death, signaling pathways that modulate this process are not known. Therefore, we used a panel of growth factors/kinase inhibitors to test effects on ferroptosis induced by our lead compound. We discovered that BMS536924, a dual inhibitor of insulin-like growth and insulin receptors, is a potent inhibitor of ferroptosis. Further investigation indicated that the anti-ferroptotic activity of BMS536924 does not lie in its ability to inhibit insulin signal transduction. Instead, we provide evidence that BMS536924 binds iron, an essential cofactor in ferroptosis. Our results suggest caution in interpreting the effects of BMS536924 in investigations of insulin signaling and uncover a novel ferroptosis inhibitor.

Discovery of Annulating Reagents Enabling the One-Step and Highly Stereoselective Synthesis of Cyclopentyl and Cyclohexyl Cores.

The use of the unprecedented annulating reagents methyl N-(tert-butylsulfinyl)-4-chlorobutanimidate and methyl N-(tert-butylsulfinyl)-5-bromopentanimidate enables the diastereoselective preparation of 5- and 6-membered carbocycles bearing three contiguous stereocenters. These synthons undergo cycloaddition with a variety of Michael acceptors to form cyclopentane/cyclohexane rings with excellent stereochemical control, generating only one of the eight possible diastereomers. This novel methodology has enabled the highly enantioselective and high yielding synthesis of novel chemotypes of pharmacological relevance.

Determination of an effective housekeeping gene for the quantification of mRNA for forensic applications.

The potential application of mRNA for the identification of biological fluids using molecular techniques has been a recent development in forensic serology. Constitutively expressed housekeeping genes can assess the amount of mRNA recovered from a sample, establish its suitability for downstream applications, and provide a reference point to corroborate the identity of the fluid. qPCR was utilized to compare the expression levels of housekeeping genes from forensic-like body fluid stains to establish the most appropriate assessment of human mRNA quantity prior to profiling. Although variability was observed between fluids and individuals, results indicated that beta-2 microglobulin exhibited the highest expression for all body fluids examined and across donors. A one-way analysis of variance was performed for housekeeping gene variability between donors (at the α, 0.05, significance level), and the results indicated significant differences for semen, vaginal secretions, and menstrual blood.