Chemical Biology Gateways to Mapping Location, Association, and Pathway Responsivity

Here we discuss, how by applying chemical concepts to biological problems, methods have been developed to map spatiotemporal regulation of proteins and small-molecule modulation of proteome signaling responses. We outline why chemical-biology platforms are ideal for such purposes. We further discuss strengths and weaknesses of chemical-biology protocols, contrasting them against classical genetic and biochemical approaches. We make these evaluations based on three parameters: occupancy; functional information; and spatial restriction. We demonstrate how the specific choice of chemical reagent and experimental set-up unite to resolve biological problems. Potential improvements/extensions as well as specific controls that in our opinion are often overlooked or employed incorrectly are also considered. Finally, we discuss some of the latest emerging methods to illuminate how chemical-biology innovations provide a gateway toward information hitherto inaccessible by conventional genetic/biochemical means. Finally, we also caution against solely relying on chemical-biology strategies and urge the field to undertake orthogonal validations to ensure robustness of results

Clofarabine Commandeers the RNR-alpha-ZRANB3 Nuclear Signaling Axis

Ribonucleotide reductase (RNR) is an essential enzyme in DNA biogenesis and a target of several chemotherapeutics. Here, we investigate how antileukemic drugs (e.g., clofarabine [CIF]) that target one of the two subunits of RNR, RNR-alpha, affect noncanonical RNR-alpha functions. We discovered that these clinically approved RNR-inhibiting dATP-analogs inhibit growth by also targeting ZRANB3-a recently identified DNA synthesis promoter and nuclear-localized interactor of RNR-alpha. Remarkably, in early time points following drug treatment, ZRANB3 targeting accounted for most of the drug-induced DNA synthesis suppression and multiple cell types featuring ZRANB3 knockout/knockdown were resistant to these drugs. In addition, ZRANB3 plays a major role in regulating tumor invasion and H-ras(G12V)-promoted transformation in a manner dependent on the recently discovered interactome of RNR-alpha involving select cytosolic-/nuclear-localized protein players. The H-ras(G12V)-promoted transformation-which we show requires ZRANB3-supported DNA synthesis-was efficiently suppressed by ClF. Such overlookedmechanisms of action of approved drugs and a previously unappreciated example of non-oncogene addiction, which is suppressed by RNR-alpha, may advance cancer interventions

Can Precision Electrophile Signaling Make a Meaningful and Lasting Impression in Drug Design?

For several years, drugs with reactive electrophilic appendages have been developed. These units typically confer prolonged residence time of the drugs on their protein targets, and may assist targeting shallow binding sites and/or improving the drug-protein target spectrum. Studies on natural electrophilic molecules have indicated that, in many instances, natural electrophiles use similar mechanisms to alter signaling pathways. However, natural reactive species are also endowed with other important mechanisms to hone signaling properties that are uncommon in drug design. These include ability to be active at low occupancy and elevated inhibitor kinetics. Herein, we discuss how we have begun to harness these properties in inhibitor design

Keap 1: The new Janus word on the block

Here we draw insights from the latest serendipitous findings made on the opposing roles of a proposed drug-target protein Keap1. We weigh up how natural reactive electrophiles and electrophilic small-molecule drugs in clinical use directly impinge on seemingly conflicting, yet both Keap1-electrophile-modification-dependent, cell -survival-vs. cell-death-promoting behaviors. In the process, we convey how understanding reactive chemical-signal regulation at the single-protein-specific level is an enabling necessity in deconstructing otherwise intricate reactive-small-molecule-responsive cellular pathways. We hope this opinion piece further spurs the broader interests of basic and pharmaceutical research communities toward better understanding of molecular mechanisms underpinning reactive small-molecule-regulated signaling subsystems

Neighborhood watch: tools for defining locale-dependent subproteomes and their contextual signaling activities

Transient associations between numerous organelles-e.g., the endoplasmic reticulum and the mitochondria-forge highly-coordinated, particular environments essential for cross-compartment information flow. Our perspective summarizes chemical-biology tools that have enabled identifying proteins present within these itinerant communities against the bulk proteome, even when a particular protein's presence is fleeting/substoichiometric. However, proteins resident at these ephemeral junctions also experience transitory changes to their interactomes, small-molecule signalomes, and, importantly, functions. Thus, a thorough census of sub-organellar communities necessitates functionally probing context-dependent signaling properties of individual protein-players. Our perspective accordingly further discusses how repurposing of existing tools could allow us to glean a functional understanding of protein-specific signaling activities altered as a result of organelles pulling together. Collectively, our perspective strives to usher new chemical-biology techniques that could, in turn, open doors to modulate functions of specific subproteomes/organellar junctions underlying the nuanced regulatory subsystem broadly termed as contactology

Getting the Right Grip? How Understanding Electrophile Selectivity Profiles Could Illuminate Our Understanding of Redox Signaling

Recent Advances: Herein, we focus on the first step of this process, electrophile sensing. Electrophile sensing is typically a deceptively simple reaction between the thiol of a protein cysteine, of which there are around 200,000 in the human proteome, and a Michael acceptor, of which there are numerous flavors, including enals and enones. Recent data overall paint a picture that despite being a simple chemical reaction, electrophile sensing is a discerning process, showing labeling preferences that are often not in line with reactivity of the electrophile. Critical Issues: With a view to trying to decide what brings about highly electrophile-reactive protein cysteines, and how reactive these sensors may be, we discuss aspects of the thermodynamics and kinetics of covalent/noncovalent binding. Data made available by several laboratories indicate that it is likely that specific proteins exhibit highly stereo- and chemoselective electrophile sensing, which we take as good evidence for recognition between the electrophile and the protein before forming a covalent bond. Future Directions: We propose experiments that could help us gain a better and more quantitative understanding of the mechanisms through which sensing comes about. We further extoll the importance of performing more detailed experiments on labeling and trying to standardize the way we assess protein-specific electrophile sensing

An Oculus to Profile and Probe Target Engagement In Vivo: How T-REX Was Born and Its Evolution into G-REX

Here we provide a personal account of innovation and design principles underpinning a method to interrogate precision electrophile signaling that has come to be known as "REX technologies". This Account is framed in the context of trying to improve methods of target mining and understanding of individual target-ligand engagement by a specific natural electrophile and the ramifications of this labeling event in cells and organisms. We start by explaining from a practical standpoint why gleaning such understanding is critical: we are constantly assailed by a battery of electrophilic molecules that exist as a consequence of diet, food preparation, ineluctable endogenous metabolic processes, and potentially disease. The resulting molecules, which are detectable in the body, appear to be able to modify function of specific proteins. Aside from potentially being biologically relevant in their own right, these labeling events are essentially identical to protein-covalent drug interactions. Thus, on what proteins and even in what ways a covalent drug will work can be understood through the eyes of natural electrophiles; extending this logic leads to the postulate that target identification of specific electrophiles can inform on drug design. However, when we entered this field, there was no way to interrogate how a specific labeling event impacted a specific protein in an unperturbed cell. Methods to evaluate stoichiometry of labeling, and even chemospecificity of a specific phenotype were limited. There were further no generally accepted ways to study electrophile signaling that did not hugely disturb physiology.We developed T-REX, a method to study single-protein-specific electrophile engagement, to interrogate how single-protein electrophile labeling shapes pathway flux. Using T-REX, we discovered that labeling of several proteins by a specific electrophile, even at low occupancy, leads to biologically relevant signaling outputs. Further experimentation using T-REX showed that in some instances, single-protein isoforms were electrophile responsive against other isoforms, such as Akt3. Selective electrophile-labeling of Akt3 elicited inhibition of Akt-pathway flux in cells and in zebrafish embryos. Using these data, we rationally designed a molecule to selectively target Akt3. This was a fusion of the naturally derived electrophile and an isoform-nonspecific, reversible Akt inhibitor in phase-II trials, MK-2206. The resulting molecule was a selective inhibitor of Akt3 and was shown to fare better than MK-2206 in breast cancer xenograft mouse models. Recently, we have also developed a means to screen electrophile sensors that is unbiased and uses a precise burst of electrophiles. Using this method, dubbed G-REX, in conjunction with T-REX, we discovered new DNA-damage response upregulation pathways orchestrated by simple natural electrophiles. We thus emphasize how deriving a quantitative understanding of electrophile signaling that is linked to thorough and precise mechanistic studies can open doors to numerous medicinally and biologically relevant insights, from gleaning better understanding of target engagement and target mining to rational design of targeted covalent medicines

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

Proteomics and Beyond: Cell Decision-Making Shaped by Reactive Electrophiles

Revolutionary proteomic strategies have enabled rapid profiling of the cellular targets of electrophilic small molecules. However, precise means to directly interrogate how these individual electrophilic modifications at low occupancy functionally reshape signaling networks have until recently been largely limited. We highlight here new methods that transcend proteomic platforms to forge a quantitative link between protein target-selective engagement and downstream signaling. We focus on recent progress in the study of non-enzyme-assisted signaling mechanisms and crosstalk choreographed by native reactive electrophilic species (RES). Using this as a model, we offer a long-term vision of how these toolsets together with fundamental biochemical knowledge of precision electrophile signaling may be harnessed to assist covalent ligand-target matching and ultimately amend disease-specific signaling dysfunction