Monitoring On-Target Signaling Responses in Larval Zebrafish - Z-REX Unmasks Precise Mechanisms of Electrophilic Drugs and Metabolites.

Reactive metabolites and related electrophilic drugs are among the most challenging small molecules to study. Conventional approaches to deconstruct the mode of action (MOA) of such molecules leverage bulk treatment of experimental specimens with an excess of a specific reactive species. In this approach, the high reactivity of electrophiles renders non-discriminate labeling of the proteome in a time- and context-dependent manner; redox-sensitive proteins and processes can also be indirectly and often irreversibly affected. Against such a backdrop of innumerable potential targets and indirect secondary effects, linking phenotype to specific target engagement remains a complex task. Zebrafish targeting reactive electrophiles and oxidants (Z-REX)-an on-demand reactive-electrophile delivery platform adapted for use in larval zebrafish-is designed to deliver electrophiles to a specific protein of interest (POI) in otherwise unperturbed live fish embryos. Key features of this technique include a low level of invasiveness, along with dosage-, chemotype-, and spatiotemporally-controlled precision electrophile delivery. Thus, in conjunction with a unique suite of controls, this technique sidesteps off-target effects and systemic toxicity, otherwise observed following uncontrolled bulk exposure of animals to reactive electrophiles and pleiotropic electrophilic drugs. Leveraging Z-REX, researchers can establish a foothold in the understanding of how individual stress responses and signaling outputs are altered as a result of specific reactive ligand engagement with a specific POI, under near-physiologic conditions in intact living animals.

Still no Rest for the Reductases: Ribonucleotide Reductase (RNR) Structure and Function: An Update.

Herein we present a multidisciplinary discussion of ribonucleotide reductase (RNR), the essential enzyme uniquely responsible for conversion of ribonucleotides to deoxyribonucleotides. This chapter primarily presents an overview of this multifaceted and complex enzyme, covering RNR's role in enzymology, biochemistry, medicinal chemistry, and cell biology. It further focuses on RNR from mammals, whose interesting and often conflicting roles in health and disease are coming more into focus. We present pitfalls that we think have not always been dealt with by researchers in each area and further seek to unite some of the field-specific observations surrounding this enzyme. Our work is thus not intended to cover any one topic in extreme detail, but rather give what we consider to be the necessary broad grounding to understand this critical enzyme holistically. Although this is an approach we have advocated in many different areas of scientific research, there is arguably no other single enzyme that embodies the need for such broad study than RNR. Thus, we submit that RNR itself is a paradigm of interdisciplinary research that is of interest from the perspective of the generalist and the specialist alike. We hope that the discussions herein will thus be helpful to not only those wanting to tackle RNR-specific problems, but also those working on similar interdisciplinary projects centering around other enzymes.

A primer on harnessing non-enzymatic post-translational modifications for drug design.

Of the manifold concepts in drug discovery and design, covalent drugs have re-emerged as one of the most promising over the past 20-or so years. All such drugs harness the ability of a covalent bond to drive an interaction between a target biomolecule, typically a protein, and a small molecule. Formation of a covalent bond necessarily prolongs target engagement, opening avenues to targeting shallower binding sites, protein complexes, and other difficult to drug manifolds, amongst other virtues. This opinion piece discusses frameworks around which to develop covalent drugs. Our argument, based on results from our research program on natural electrophile signaling, is that targeting specific residues innately involved in native signaling programs are ideally poised to be targeted by covalent drugs. We outline ways to identify electrophile-sensing residues, and discuss how studying ramifications of innate signaling by endogenous molecules can provide a means to predict drug mechanism and function and assess on- versus off-target behaviors.

Understanding the enzyme-ligand complex: insights from all-atom simulations of butyrylcholinesterase inhibition.

All-atom molecular dynamics simulations of butyrylcholinesterase (BChE) sans inhibitor and in complex with each of 15 dialkyl phenyl phosphate derivatives were conducted to characterize inhibitor binding modes and strengths. Each system was sampled on the 250 ns timescale in explicit ionic solvent, for a total of over 4 µs of simulation time. A K-means algorithm was used to cluster the resulting structures into distinct binding modes, which were further characterized based on atomic-level contacts between inhibitor chemical groups and active site residues. Comparison of experimentally observed inhibition constants (KI) with the resulting contact tables provides structural explanations for relative binding coefficients and highlights several notable interaction motifs. These include ubiquitous contact between glycines in the oxyanion hole and the inhibitor phosphate group; a sterically driven binding preference for positional isomers that extend aromaticity; a stereochemical binding preference for choline-containing inhibitors, which mimic natural BChE substrates; and the mechanically induced opening of the omega loop region to fully expose the active site gorge in the presence of choline-containing inhibitors. Taken together, these observations can greatly inform future design of BChE inhibitors, and the approach reported herein is generalizable to other enzyme-inhibitor systems and similar complexes that depend on non-covalent molecular recognition.Communicated by Ramaswamy H. Sarma.