Regenerative medicine for heart failure seeks to replace lost cardiomyocytes. Chemical approaches for producing ample supplies of cells, such as pluripotent stem cells and cardiomyocytes, hold promise as practical means to achieve safe, facile cell-based therapy for cardiac repair and regenerative medicine. In this review, we describe recent advances in the application of small molecules to improve the generation and maintenance of pluripotent stem cells. We also describe new directions in heart repair and regeneration in which chemical approaches may find their application.

Understanding the processes by which stem cells give rise to de novo tissues is an active focus of stem cell biology and bioengineering disciplines. Instructive morphogenic cues surrounding the stem cell during morphogenesis create what is referred to as the stem cell microenvironment. An emerging paradigm in stem cell bioengineering involves “biologically driven assembly,” in which stem cells are encouraged to largely define their own morphogenesis processes. However, even in the case of biologically driven assembly, stem cells do not act alone. The properties of the surrounding microenvironment can be critical regulators of cell fate. Stem cell–material interactions are among the most well-characterized microenvironmental effectors of stem cell fate and establish a signaling “context” that can define the mode of influence for morphogenic cues. Here we describe illustrative examples of cell–material interactions that occur during in vitro stem cell studies, with an emphasis on how cell–material interactions create instructive contexts for stem cell differentiation and morphogenesis.

The field of regenerative medicine has boomed in recent years thanks to milestone discoveries in stem cell biology and tissue engineering, which has been driving paradigm shifts in the pharmacotherapy of degenerative and ischemic diseases. Small molecule-mediated replenishment of lost and/or dysfunctional tissue in vivo, however, is still in its infancy due to a limited understanding of mechanisms that control such endogenous processes of tissue homeostasis or regeneration. Here, we discuss current progress using small molecules targeting in vivo aspects of regeneration, including adult stem cells, stem cell niches, and mechanisms of homing, mobilization, and engraftment as well as somatic cell proliferation. Many of these compounds derived from both knowledge-based design and screening campaigns, illustrating the feasibility of translating in vitro discovery to in vivo regeneration. These early examples of drug-mediated in vivo regeneration provide a glimpse of the future directions of in vivo regenerative medicine approaches.

Repairing damaged cartilage and tendons is a major challenge of regenerative medicine. There has been great progress in the past decade toward obtaining stem cells for regenerative purposes from a variety of sources. However, the development of procedures to direct and maintain the differentiation of progenitors into cartilage or tendon is still a hurdle to overcome in regenerative medicine of the musculoskeletal system. This is because connective tissues often lack stable phenotypes and retain plasticity to return to the initial stages of differentiation or to transdifferentiate into another connective tissue cell lineage. This makes it necessary to unravel the molecular basis that is responsible for the differentiation of connective tissue cell lineages. In this review, we summarize the investigations performed in the past two decades to unravel the signals that regulate the differentiation of skeletal cell progenitors into cartilage and tendons during embryonic limb development. The data obtained in those studies demonstrate that Tgfβ, BMP, FGF, and Wnt establish a complex signaling network that directs the differentiation of skeletal cell progenitors. Remarkably, in the embryonic digit model, the divergent differentiation of progenitors depends on the temporal coordination of those signals, rather than being specified by an individual signaling pathway. Due to its potential medical relevance, we highlight the importance of the coordinate influence of the Tgfβ and BMP pathways in the differentiation of cell progenitors into tendon or cartilage.

Stem cell transplantation is a potential therapy for regenerative medicine, which aims to restore tissues damaged by trauma, aging, and diseases. Since its conception in the late 1990s, chemical biology has provided powerful and diverse small molecule tools for modulating stem cell function. Embryonic stem cells could be an ideal source for transplantation, but ethical concerns restrict their development for cell therapy. The seminal advance of induced pluripotent stem cell (iPSC) technology provided an attractive alternative to human embryonic stem cells. However, iPSCs are not yet considered an ideal stem cell source, due to limitations associated with the reprogramming process and their potential tumorigenic behavior. This is an area of research where chemical biology has made a significant contribution to facilitate the efficient production of high quality iPSCs and elucidate the biological mechanisms governing their phenotype. In this review, we summarize these advances and discuss the latest progress in developing small molecule modulators. Moreover, we also review a new trend in stem cell research, which is the direct reprogramming of readily accessible cell types into clinically useful cells, such as neurons and cardiac cells. This is a research area where chemical biology is making a pivotal contribution and illustrates the many advantages of using small molecules in stem cell research.

Glycans participate in many key cellular processes during development and in physiology and disease. In this review, the functional role of various glycans in the regeneration of neurons and body parts in adult metazoans is discussed. Understanding glycosylation may facilitate research in the field of stem cell biology and regenerative medicine.

A complication of diabetes is the inability of wounds to heal in diabetic patients. Diabetic wounds are refractory to healing due to the involvement of activated matrix metalloproteinases (MMPs), which remodel the tissue resulting in apoptosis. There are no readily available methods that identify active unregulated MMPs. With the use of a novel inhibitor-tethered resin that binds exclusively to the active forms of MMPs, coupled with proteomics, we quantified MMP-8 and MMP-9 in a mouse model of diabetic wounds. Topical treatment with a selective MMP-9 inhibitor led to acceleration of wound healing, re-epithelialization, and significantly attenuated apoptosis. In contrast, selective pharmacological inhibition of MMP-8 delayed wound healing, decreased re-epithelialization, and exhibited high apoptosis. The MMP-9 activity makes the wounds refractory to healing, whereas that of MMP-8 is beneficial. The treatment of diabetic wounds with a selective MMP-9 inhibitor holds great promise in providing heretofore-unavailable opportunities for intervention of this disease.

Post-translational regulation of protein abundance in cells is a powerful tool for studying protein function. Here, we describe a novel genetically encoded protein domain that is degraded upon exposure to nontoxic blue light. We demonstrate that fusion proteins containing this domain are rapidly degraded in cultured cells and in zebrafish upon illumination.

Metal selectivity in P1B-type ATPase transporters is determined by conserved amino acid residues in their transmembrane helices responsible for metal binding and transport across the cellular membrane. The Cu2+-selective CopB from Archaeoglobus fulgidus has been investigated to explore the coordination chemistry of the transition metal binding sites in P1B-3-type ATPases. Electronic absorption, electron paramagnetic resonance, and X-ray absorption spectroscopic studies indicate the presence of a high-affinity transmembrane Type-2-like Cu2+ center in which a single cupric ion is coordinated in a distorted square pyramidal geometry by mixed nitrogen/oxygen and sulfur ligands.

Infections caused by multi-drug resistant bacteria, particularly Gram-negative bacteria, are an ever-increasing problem. While the development of new antibiotics remains one option in the fight against bacteria that have become resistant to currently available antibiotics, an attractive alternative is the development of adjuvant therapeutics that restore the efficacy of existing antibiotics. We report a small molecule adjuvant that suppresses colistin resistance in multidrug resistant Acinetobacter baumannii and Klebsiella pneumoniae by interfering with the expression of a two-component system. The compound downregulates the pmrCAB operon and reverses phosphoethanolamine modification of lipid A responsible for colistin resistance. Furthermore, colistin-susceptible and colistin-resistant bacteria do not evolve resistance to combination treatment. This represents the first definitive example of a compound that breaks antibiotic resistance by directly modulating two-component system activity.

Protein dimerization provides a mechanism for the modulation of cellular signaling events. In α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors, the rapidly desensitizing, activated state has been correlated with a weakly dimeric, glutamate-binding domain conformation. Allosteric modulators can form bridging interactions that stabilize the dimer interface. While most modulators can only bind to one position with a one modulator per dimer ratio, some thiazide-based modulators can bind to the interface in two symmetrical positions with a two modulator per dimer ratio. Based on small-angle X-ray scattering (SAXS) experiments, dimerization curves for the isolated glutamate-binding domain show that a second modulator binding site produces both an increase in positive cooperativity and a decrease in the EC50 for dimerization. Four body binding equilibrium models that incorporate a second dimer-stabilizing ligand were developed to fit the experimental data. The work illustrates why stoichiometry should be an important consideration during the rational design of dimerizing modulators.

Genetically engineered reporters have revolutionized the understanding of many biological processes. MRI-based reporter genes can dramatically improve our ability to monitor dynamic gene expression and allow coregistration of subcellular genetic information with high-resolution anatomical images. We have developed a biocompatible MRI reporter gene based on a human gene, the human protamine-1 (hPRM1). The arginine-rich hPRM1 (47% arginine residues) generates high MRI contrast based on the chemical exchange saturation transfer (CEST) contrast mechanism. The 51 amino acid-long hPRM1 protein was fully synthesized using microwave-assisted technology, and the CEST characteristics of this protein were compared to other CEST-based contrast agents. Both bacterial and human cells were engineered to express an optimized hPRM1 gene and showed higher CEST contrast compared to controls. Live cells expressing the hPRM1 reporter gene, and embedded in three-dimensional culture, also generated higher CEST contrast compared to wild-type live cells.

Embryonic stem (ES) cells are derived from the inner cell mass of the blastocyst stage embryo and are characterized by self-renewal and pluripotency. Previous work has shown that Src-family tyrosine kinases display dynamic expression and activity changes during ES cell differentiation, suggesting distinct functions in the control of developmental fate. Here we used ES cells to test the hypothesis that c-Src and its closest phylogenetic relative, c-Yes, act in biological opposition despite their strong homology. Unlike c-Src, enforced expression of active c-Yes blocked ES cell differentiation to embryoid bodies by maintaining pluripotency gene expression. To explore the interplay of c-Src and c-Yes in ES cell differentiation, we engineered c-Src and c-Yes mutants that are resistant to A-419259, a potent pyrrolopyrimidine inhibitor of the Src kinase family. Previous studies have shown that A-419259 treatment blocks all Src-family kinase activity in ES cells, preventing differentiation while maintaining pluripotency. Expression of inhibitor-resistant c-Src but not c-Yes rescued the A-419259 differentiation block, resulting in a cell population with properties of both primitive ectoderm and endoderm. Remarkably, when inhibitor-resistant c-Src and c-Yes were expressed together in ES cells, c-Yes activity suppressed c-Src-mediated differentiation. These studies show that even closely related kinases such as c-Src and c-Yes have unique and opposing functions in the same cell type. Selective agonists or inhibitors of c-Src versus c-Yes activity may allow more precise pharmacological manipulation of ES cell fate and have broader applications in other biological systems that express multiple Src family members such as tumor cells.

Cell–cell communications, cell–matrix interactions, and cell migrations play a major role in regeneration. However, little is known about the molecular players involved in these critical events, especially cell surface molecules. Here, we demonstrate the role of specific glycan–receptor interactions in the regenerative process using Hydra magnipapillata as a model system. Global characterization of the N- and O-glycans expressed by H. magnipapillata using ultrasensitive mass spectrometry revealed mainly polyfucosylated LacdiNAc antennary structures. Affinity purification showed that a putative C-type lectin (accession number Q6SIX6) is a likely endogenous receptor for the novel polyfucosylated glycans. Disruption of glycan–receptor interactions led to complete shutdown of the regeneration machinery in live Hydra. A time-dependent, lack-of-regeneration phenotype observed upon incubation with exogenous fuco-lectins suggests the involvement of a polyfucose receptor-mediated signaling mechanism during regeneration. Thus, for the first time, the results presented here provide direct evidence for the role of polyfucosylated glycan–receptor interactions in the regeneration of H. magnipapillata.

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS) and is characterized by the destruction of myelin and axons leading to progressive disability. Peptide epitopes from CNS proteins, such as myelin oligodendrocyte glycoprotein (MOG), possess promising immunoregulatory potential for treating MS; however, their instability and poor bioavailability is a major impediment for their use clinically. To overcome this problem, we used molecular grafting to incorporate peptide sequences from the MOG35–55 epitope onto a cyclotide, which is a macrocyclic peptide scaffold that has been shown to be intrinsically stable. Using this approach, we designed novel cyclic peptides that retained the structure and stability of the parent scaffold. One of the grafted peptides, MOG3, displayed potent ability to prevent disease development in a mouse model of MS. These results demonstrate the potential of bioengineered cyclic peptides for the treatment of MS.

The sesquiterpene lactone parthenolide has recently attracted considerable attention owing to its promising antitumor properties, in particular in the context of stem-cell cancers including leukemia. Yet, the lack of viable synthetic routes for re-elaborating this complex natural product has represented a fundamental obstacle toward further optimization of its pharmacological properties. Here, we demonstrate how this challenge could be addressed via selective, late-stage sp3 C–H bond functionalization mediated by P450 catalysts with tailored site-selectivity. Taking advantage of our recently introduced tools for high-throughput P450 fingerprinting and fingerprint-driven P450 reactivity prediction, we evolved P450 variants useful for carrying out the highly regioselective hydroxylation of two aliphatic sites (C9 and C14) in parthenolide carbocyclic backbone. By chemoenzymatic synthesis, a panel of novel C9- and C14-modified parthenolide analogs were generated in order to gain initial structure–activity insights on these previously inaccessible sites of the molecule. Notably, some of these compounds were found to possess significantly improved antileukemic potency against primary acute myeloid leukemia cells, while exhibiting low toxicity against normal mature and progenitor hematopoietic cells. By identifying two ‘hot spots’ for improving the anticancer properties of parthenolide, this study highlights the potential of P450-mediated C–H functionalization as an enabling, new strategy for the late-stage manipulation of bioactive natural product scaffolds.

RNA aptamers, selected from large synthetic libraries, are attracting increasing interest as protein ligands, with potential uses as prototype pharmaceuticals, conformational probes, and reagents for specific quantification of protein levels in biological samples. Very little is known, however, about their effects on protein conformation and dynamics. We have employed hydrogen/deuterium exchange (HDX) mass spectrometry to study the effect of RNA aptamers on the structural flexibility of the serpin plasminogen activator inhibitor-1 (PAI-1). The aptamers have characteristic effects on the biochemical properties of PAI-1. In particular, they are potent inhibitors of the structural transition of PAI-1 from the active state to the inactive, so-called latent state. This transition is one of the largest conformational changes of a folded protein domain without covalent modification. Binding of the aptamers to PAI-1 is associated with substantial and widespread protection against deuterium uptake in PAI-1. The aptamers induce protection against exchange with the solvent both in the protein-aptamer interface as well as in other specific areas. Interestingly, the aptamers induce substantial protection against exchange in α-helices B, C and I. This observation substantiates the relevance of structural instability in this region for transition to the latent state and argues for involvement of flexibility in regions not commonly associated with regulation of latency transition in serpins.

The rise of bacterial resistance to traditional antibiotics has motivated recent efforts to identify new drug candidates that target virulence factors or their regulatory pathways. One such antivirulence target is the cyclic-di-GMP (cdiGMP) signaling pathway, which regulates biofilm formation, motility, and pathogenesis. Pseudomonas aeruginosa is an important opportunistic pathogen that utilizes cdiGMP-regulated polysaccharides, including alginate and pellicle polysaccharide (PEL), to mediate virulence and antibiotic resistance. CdiGMP activates PEL and alginate biosynthesis by binding to specific receptors including PelD and Alg44. Mutations that abrogate cdiGMP binding to these receptors prevent polysaccharide production. Identification of small molecules that can inhibit cdiGMP binding to the allosteric sites on these proteins could mimic binding defective mutants and potentially reduce biofilm formation or alginate secretion. Here, we report the development of a rapid and quantitative high-throughput screen for inhibitors of protein-cdiGMP interactions based on the differential radial capillary action of ligand assay (DRaCALA). Using this approach, we identified ebselen as an inhibitor of cdiGMP binding to receptors containing an RxxD domain including PelD and diguanylate cyclases (DGC). Ebselen reduces diguanylate cyclase activity by covalently modifying cysteine residues. Ebselen oxide, the selenone analogue of ebselen, also inhibits cdiGMP binding through the same covalent mechanism. Ebselen and ebselen oxide inhibit cdiGMP regulation of biofilm formation and flagella-mediated motility in P. aeruginosa through inhibition of diguanylate cyclases. The identification of ebselen provides a proof-of-principle that a DRaCALA high-throughput screening approach can be used to identify bioactive agents that reverse regulation of cdiGMP signaling by targeting cdiGMP-binding domains.

Selective disruption of protein–protein interactions by small molecules is important for probing the structure and dynamic aspects of cellular network. It can also provide new therapeutic targets. β-Catenin of the canonical Wnt signaling pathway uses the same positively charged groove to bind with T-cell factor (Tcf), cadherin, and adenomatous polysis coli (APC). The extravagant formation of β-catenin/Tcf interactions drives the initiation and progression of many cancers and fibroses, while β-catenin/cadherin and β-catenin/APC interactions are essential for cell–cell adhesion and β-catenin degradation. In this study, a selective binding site that can differentiate β-catenin/Tcf, β-catenin/cadherin, and β-catenin/APC interactions was identified by alanine scanning and biochemical assays. A new peptidomimetic strategy that incorporates SiteMap and multiple-copy simultaneous search was used to design selective small-molecule inhibitors for β-catenin/Tcf interactions. A potent inhibitor was discovered to bind with β-catenin and completely disrupt β-catenin/Tcf interactions. It also exhibits dual selectivity for β-catenin/Tcf over β-catenin/cadherin and β-catenin/APC interactions in both biochemical and cell-based assays. This study provides a proof of concept for designing selective inhibitors for β-catenin/Tcf interactions.

B cells detect foreign antigens through their B cell antigen receptor (BCR). The BCR, when engaged by antigen, initiates a signaling cascade. Concurrent with signaling is endocytosis of the BCR complex, which acts to downregulate signaling and facilitate uptake of antigen for processing and display on the cell surface. The relationship between signaling and BCR endocytosis is poorly defined. Here, we explore the interplay between BCR endocytosis and antigens that either promote or inhibit B cell activation. Specifically, synthetic antigens were generated that engage the BCR alone or both the BCR and the inhibitory co-receptor CD22. The lectin CD22, a member of the Siglec family, binds sialic acid-containing glycoconjugates found on host tissues, inhibiting BCR signaling to prevent erroneous B cell activation. At low concentrations, antigens that can cocluster the BCR and CD22 promote rapid BCR endocytosis; whereas, slower endocytosis occurs with antigens that bind only the BCR. At higher antigen concentrations, rapid BCR endocytosis occurs upon treatment with either stimulatory or inhibitory antigens. Endocytosis of the BCR, in response to synthetic antigens, results in its entry into early endocytic compartments. Although the CD22-binding antigens fail to activate key regulators of antigen presentation (e.g., Syk), they also promote BCR endocytosis, indicating that inhibitory antigens can be internalized. Together, our observations support a functional role for BCR endocytosis in downregulating BCR signaling. The reduction of cell surface BCR levels in the absence of B cell activation should raise the threshold for BCR subsequent activation. The ability of the activating synthetic antigens to trigger both signaling and entry of the BCR into early endosomes suggests strategies for targeted antigen delivery.

Antimicrobial peptides (AMPs) are host-defense agents capable of both bacterial membrane disruption and immunomodulation. However, the development of natural AMPs as potential therapeutics is hampered by their moderate activity and susceptibility to protease degradation. Herein we report lipidated cyclic γ-AApeptides that have potent antibacterial activity against clinically relevant Gram-positive and Gram-negative bacteria, many of which are resistant to conventional antibiotics. We show that lipidated cyclic γ-AApeptides mimic the bactericidal mechanism of AMPs by disrupting bacterial membranes. Interestingly, they also harness the immune response and inhibit lipopolysaccharide (LPS) activated Toll-like receptor 4 (TLR4) signaling, suggesting that lipidated cyclic γ-AApeptides have dual roles as novel antimicrobial and anti-inflammatory agents.

This study highlights the benefits of nano electrospray ionization mass spectrometry (nanoESI-MS) as a fast and label-free method not only for determination of dissociation constants (KD) of a cooperatively regulated enzyme but also to better understand the mechanism of enzymatic cooperativity of multimeric proteins. We present an approach to investigate the allosteric mechanism in the binding of inhibitors to the homotetrameric enzyme fructose 1,6-bisphosphatase (FBPase), a potential therapeutic target for glucose control in type 2 diabetes. A series of inhibitors binding at an allosteric site of FBPase were investigated to determine their KDs by nanoESI-MS. The KDs determined by ESI-MS correlate very well with IC50 values in solution. The Hill coefficients derived from nanoESI-MS suggest positive cooperativity. From single-point measurements we could obtain information on relative potency, stoichiometry, conformational changes, and mechanism of cooperativity. A new X-ray crystal structure of FBPase tetramer binding ligand 3 in a 4:4 stoichiometry is also reported. NanoESI-MS-based results match the current understanding of the investigated system and are in agreement with the X-ray structural data, but provide additional mechanistic insight on the ligand binding, due to the better dynamic resolution. This method offers a powerful approach for studying other proteins with allosteric binding sites, as well.

Structural diversification of canonical nucleic acid bases and nucleotide analogues by tautomerism has been proposed to be a powerful on/off switching mechanism allowing regulation of many biological processes mediated by RNA enzymes and aptamers. Despite the suspected biological importance of tautomerism, attempts to observe minor tautomeric forms in nucleic acid or hybrid nucleic acid-ligand complexes have met with challenges due to the lack of sensitive methods. Here, a combination of spectroscopic, biochemical, and computational tools probed tautomerism in the context of an RNA aptamer-ligand complex; studies involved a model ligand, oxythiamine pyrophosphate (OxyTPP), bound to the thiamine pyrophosphate (TPP) riboswitch (an RNA aptamer) as well as its unbound nonphosphorylated form, oxythiamine (OxyT). OxyTPP, similarly to canonical heteroaromatic nucleic acid bases, has a pyrimidine ring that forms hydrogen bonding interactions with the riboswitch. Tautomerism was established using two-dimensional infrared (2D IR) spectroscopy, variable temperature FTIR and NMR spectroscopies, binding isotope effects (BIEs), and computational methods. All three possible tautomers of OxyT, including the minor enol tautomer, were directly identified, and their distributions were quantitated. In the bound form, BIE data suggested that OxyTPP existed as a 4′-keto tautomer that was likely protonated at the N1′-position. These results also provide a mechanistic framework for understanding the activation of riboswitch in response to deamination of the active form of vitamin B1 (or TPP). The combination of methods reported here revealing the fine details of tautomerism can be applied to other systems where the importance of tautomerism is suspected.

The LpxC enzyme in the lipid A biosynthetic pathway is one of the most promising and clinically unexploited antibiotic targets for treatment of multidrug-resistant Gram-negative infections. Progress in medicinal chemistry has led to the discovery of potent LpxC inhibitors with a variety of chemical scaffolds and distinct antibiotic profiles. The vast majority of these compounds, including the nanomolar inhibitors L-161,240 and BB-78485, are highly effective in suppressing the activity of Escherichia coli LpxC (EcLpxC) but not divergent orthologs such as Pseudomonas aeruginosa LpxC (PaLpxC) in vitro. The molecular basis for such promiscuous inhibition of EcLpxC has remained poorly understood. Here, we report the crystal structure of EcLpxC bound to L-161,240, providing the first molecular insight into L-161,240 inhibition. Additionally, structural analysis of the EcLpxC/L-161,240 complex together with the EcLpxC/BB-78485 complex reveals an unexpected backbone flipping of the Insert I βa−βb loop in EcLpxC in comparison with previously reported crystal structures of EcLpxC complexes with l-threonyl-hydroxamate-based broad-spectrum inhibitors. Such a conformational switch, which has only been observed in EcLpxC but not in divergent orthologs such as PaLpxC, results in expansion of the active site of EcLpxC, enabling it to accommodate LpxC inhibitors with a variety of head groups, including compounds containing single (R- or S-enantiomers) or double substitutions at the neighboring Cα atom of the hydroxamate warhead group. These results highlight the importance of understanding inherent conformational plasticity of target proteins in lead optimization.

Toll-like receptors (TLRs) play a critical role in innate immunity, but activation of TLR signaling pathways is also associated with many harmful inflammatory diseases. Identification of novel anti-inflammatory molecules targeting TLR signaling pathways is central to the development of new treatment approaches for acute and chronic inflammation. We performed high-throughput screening from crude marine sponge extracts on TLR5 signaling and identified girolline. We demonstrated that girolline inhibits signaling through both MyD88-dependent and -independent TLRs (i.e., TLR2, 3, 4, 5, and 7) and reduces cytokine (IL-6 and IL-8) production in human peripheral blood mononuclear cells and macrophages. Using a chemical genomics approach, we identified Elongation Factor 2 as the molecular target of girolline, which inhibits protein synthesis at the elongation step. Together these data identify the sponge natural product girolline as a potential anti-inflammatory agent acting through inhibition of protein synthesis.

Zinc ions (Zn2+) play vital catalytic, structural, and regulatory roles in protein function and are commonly chelated to cysteine residues within the protein framework. Current methods to identify Zn2+-binding cysteines rely on computational studies based on known Zn2+-chelating motifs, as well as high-resolution structural data. These available approaches preclude the global identification of putative Zn2+-chelating cysteines, particularly on poorly characterized proteins in the proteome. Herein, we describe an experimental platform that identifies metal-binding cysteines on the basis of their reduced nucleophilicity upon treatment with metal ions. As validation of our platform, we utilize a peptide-based cysteine-reactive probe to show that the known Zn2+-chelating cysteine in sorbitol dehydrogenase (SORD) demonstrates an expected loss in nucleophilicity in the presence of Zn2+ ions and a gain in nucleophilicity upon treatment with a Zn2+ chelator. We also identified the active-site cysteine in glutathione S-transferase omega-1 (GSTO1) as a potential Zn2+-chelation site, albeit with lower metal affinity relative to SORD. Treatment of recombinant GSTO1 with Zn2+ ions results in a dose-dependent decrease in GSTO1 activity. Furthermore, we apply a promiscuous cysteine-reactive probe to globally identify putative Zn2+-binding cysteines across ∼900 cysteines in the human proteome. This proteomic study identified several well-characterized Zn2+-binding proteins, as well as numerous uncharacterized proteins from functionally distinct classes. This platform is highly versatile and provides an experimental tool that complements existing computational and structural methods to identify metal-binding cysteine residues.

Previous studies in human patients and animal models have suggested that transglutaminase 2 (TG2) is upregulated in pulmonary hypertension (PH), a phenomenon that appears to be associated with the effects of serotonin (5-hydroxytryptamine; 5-HT) in this disease. Using chemical tools to interrogate and inhibit TG2 activity in vivo, we have shown that pulmonary TG2 undergoes marked post-translational activation in a mouse model of hypoxia-induced PH. We have also identified irreversible fluorinated TG2 inhibitors that may find use as non-invasive positron emission tomography probes for diagnosis and management of this debilitating, lifelong disorder. Pharmacological inhibition of TG2 attenuated the elevated right ventricular pressure but had no effect on hypertrophy of the right ventricle of the heart. A longitudinal study of pulmonary TG2 activity in PH patients is warranted.

β-Peptide foldamers offer attractive frameworks for examining the effect of backbone flexibility on the dynamics of protein folding. Herein, we study the folding–unfolding kinetics of a β-peptide, Acid-1Y,1 which folds in aqueous solution into an octameric bundle of peptides in a conformation known as the 14-helix. Acid-1Y is comprised exclusively of β-amino acids, which differ from α-amino acids by the addition of a single methylene into the backbone. We aim to understand how the additional degree of freedom and increased backbone flexibility in the β-amino acid affect folding dynamics and to measure folding rates of this octameric β-peptide. Previously, we found that the T-jump induced relaxation kinetics of a monomeric β-peptide that forms a monomeric 14-helix occurred on the nanosecond time scale2 and were noticeably slower than a similar alanine-based α-helical peptide.3 Additionally, in comparison to similar α-helices, the relaxation rates showed a weaker dependence on temperature. Here, we find that the T-jump induced relaxation kinetics of the octameric β-peptide occurs on an even slower time scale (minutes) and the unfolding relaxation rates show a large dependence on temperature. These differences indicate that folding energy landscapes of β-peptide secondary and quaternary structure are markedly distinct from one another and also from their α-helical counterparts.

The exact biological functions of individual DNA polymerases still await clarification, and therefore appropriate reagents to probe their respective functions are required. In the present study, we report the development of a highly potent series of human DNA polymerase λ and β (pol λ and β) inhibitors based on the rhodanine scaffold. Both enzymes are involved in DNA repair and are thus considered as future drug targets. We expanded the chemical diversity of the small-molecule inhibitors arising from a high content screening and designed and synthesized 30 novel analogues. By biochemical evaluation, we discovered 23 highly active compounds against pol λ. Importantly, 10 of these small-molecules selectively inhibited pol λ and not the homologous pol β. We discovered 14 small-molecules that target pol β and found out that they are more potent than known inhibitors. We also investigated whether the discovered compounds sensitize cancer cells toward DNA-damaging reagents. Thus, we cotreated human colorectal cancer cells (Caco-2) with the small-molecule inhibitors and hydrogen peroxide or the approved drug temozolomide. Interestingly, the tested compounds sensitized Caco-2 cells to both genotoxic agents in a DNA repair pathway-dependent manner.

Protein reduction–oxidation (redox) modification is an important mechanism that allows microorganisms to sense environmental changes and initiate cellular responses. We have developed a quantitative chemical probe approach for live cell labeling and imaging of proteins that are sensitive to redox modifications. We utilize this in vivo strategy to identify 176 proteins undergoing ∼5–10-fold dynamic redox change in response to nutrient limitation and subsequent replenishment in the photoautotrophic cyanobacterium Synechococcus sp. PCC 7002. We detect redox changes in as little as 30 s after nutrient perturbation and oscillations in reduction and oxidation for 60 min following the perturbation. Many of the proteins undergoing dynamic redox transformations participate in the major components for the production (photosystems and electron transport chains) or consumption (Calvin–Benson cycle and protein synthesis) of reductant and/or energy in photosynthetic organisms. Thus, our in vivo approach reveals new redox-susceptible proteins and validates those previously identified in vitro.

The epoxyketone proteasome inhibitors are an established class of therapeutic agents for the treatment of cancer. Their unique α′,β′-epoxyketone pharmacophore allows binding to the catalytic β-subunits of the proteasome with extraordinary specificity. Here, we report the characterization of the first gene clusters for the biosynthesis of natural peptidyl-epoxyketones. The clusters for epoxomicin, the lead compound for the anticancer drug Kyprolis, and for eponemycin were identified in the actinobacterial producer strains ATCC 53904 and Streptomyces hygroscopicus ATCC 53709, respectively, using a modified protocol for Ion Torrent PGM genome sequencing. Both gene clusters code for a hybrid nonribosomal peptide synthetase/polyketide synthase multifunctional enzyme complex and homologous redox enzymes. Epoxomicin and eponemycin were heterologously produced in Streptomyces albus J1046 via whole pathway expression. Moreover, we employed mass spectral molecular networking for a new comparative metabolomics approach in a heterologous system and discovered a number of putative epoxyketone derivatives. With this study, we have definitively linked epoxyketone proteasome inhibitors and their biosynthesis genes for the first time in any organism, which will now allow for their detailed biochemical investigation.