Small Molecule Substrates Research Articles

Nanosims Tools and Probes for Imaging Intracellular Structures

In eukaryotic cells, intracellular lipid membranes act as barriers that segregate specific chemical reactions into organelles for increased biochemical efficiency. Organelle efficiency is maintained by the regulation of cholesterol and different lipid distributions within these organelles and is tightly controlled by intracellular transport and lipid metabolism. Disturbances in intracellular lipid trafficking, biosynthesis and degradation are hallmarks of fatal diseases involving an accumulation of cholesterol or lipids in endosomes and lysosomes, such as Niemann Pick Type II disease. Efforts to elucidate the mechanisms for lipid-mediated cellular (dys)function would benefit from an imaging technique that enables assessing the relative abundances of cholesterol and lipid species of interest between organelles. Towards this aim, we developed a secondary ion mass spectrometry (SIMS) approach to image the sphingolipid and cholesterol distributions within intact cells in three dimensions with a NanoSIMS instrument. Because the components of interest must produce secondary ions that contain distinctive elements or isotopes to permit their detection with a NanoSIMS instrument, organelle-specific labels must be incorporated into the cells to enable identifying specific organelles. This work describes a strategy that utilizes small protein tags and functionalized substrates that permit identify organelles of interest with NanoSIMS, and the development of data processing tools that produce images of these organelles at the proper depth in the 3D SIMS images. We created a transfected cell line that stably expresses enzymatic proteins fused to organelle-specific proteins. These proteins covalently attach to functionalized small-molecule substrates which can be detected with NanoSIMS and fluorescence microscopy. By combining these tools for organelle detection with lipid-specific metabolic isotope incorporation, semi-quantitative information concerning the abundance of cholesterol or specific lipids within organelles of interest may be obtained.

Control of RNA with quinone methide reversible acylating reagents.

Caging RNA by polyacylation (cloaking) has been developed recently as a simple and rapid method to control the function of RNAs. Previous approaches for chemical reversal of acylation (uncloaking) made use of azide reduction followed by amine cyclization, requiring ∼2-4 h for the completion of cyclization. In new studies aimed at improving reversal rates and yields, we have designed novel acylating reagents that utilize quinone methide (QM) elimination for reversal. The QM de-acylation reactions were tested with two bioorthogonally cleavable motifs, azide and vinyl ether, and their acylation and reversal efficiencies were assessed with NMR and mass spectrometry on model small-molecule substrates as well as on RNAs. Successful reversal both with phosphines and strained alkenes was documented. Among the compounds tested, the azido-QM compound A-3 displayed excellent de-acylation efficiency, with t1/2 for de-acylation of less than an hour using a phosphine trigger. To test its function in RNA caging, A-3 was successfully applied to control EGFP mRNA translation in vitro and in HeLa cells. We expect that this molecular caging strategy can serve as a valuable tool for biological investigation and control of RNAs both in vitro and in cells.

A Bioorthogonal Small Molecule Selective Polymeric "Clickase".

Synthetic polymer scaffolds may serve as gatekeepers preventing the adhesion of biomacromolecules. Herein, we use gating to develop a copper-containing single-chain nanoparticle (SCNP) catalyst as an artificial "clickase" that operates selectively on small molecules that are able to penetrate the polymeric shell. Whereas the analogous clickase with surface ammonium groups performs highly efficient copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) reactions on both alkynylated proteins and small molecule substrates, the new SCNP clickase with polyethylene glycol (PEG) groups is only active on small molecules. Further, the new SCNP resists uptake by cells allowing extracellular click chemistry to be performed. We describe two proof of principle applications that illustrate the utility of the bioorthogonal activity. First, the SCNP catalyst is able to screen for ligands that bind proteins, including proteolysis targeting chimera (PROTAC)-like molecules. Second, the nonmembrane permeable SCNP can efficiently catalyze the click reaction extracellularly, thereby enabling in situ anticancer drug synthesis and screening without the catalyst perturbing intracellular functions.

The covalent SNAP tag for protein display quantification and low-pH protein engineering

Yeast display has become an important tool for modern biotechnology with many advantages for eukaryotic protein engineering. Antibody-based peptide interactions are often used to quantify yeast surface expression (e.g., by fusing a target protein to a FLAG, Myc, polyhistidine, or other peptide tag). However, antibody-antigen interactions require high stability for accurate quantification, and conventional tag systems based on such interactions may not be compatible with a low pH environment. In this study, a SNAP tag was introduced to a yeast display platform to circumvent disadvantages of conventional antibody display tags at low pH. SNAP forms a covalent bond with its small-molecule substrate, enabling precise and pH-independent protein display tagging. We compared the SNAP tag to conventional antibody-based peptide fusion and to direct fluorescent domain fusion using antibody fragment crystallizable (Fc) gene libraries as a case study in low pH protein engineering. Our results demonstrated that covalent SNAP tags can effectively quantify protein-surface expression at low pH, enabling the enrichment of Fc variants with increased affinity at pH 6.0 to the neonatal Fc receptor (FcRn). Incorporation of a covalent SNAP tag thus overcomes disadvantages of conventional antibody-based expression tags and enables protein-engineering applications outside of physiological pH.

Metabolomics Profiling in Response to a Cold Pressor Test

The cold pressor test (CPT) has been widely used to assess both cardiovascular and hemodynamic responses to stress. Despite the broad utilization of the CPT, there have been few if any studies in which metabolomic profiling was used to study the metabolic response to such a stimulus. Metabolomics allows insight into varying chemical processes, displaying detailed profiles of metabolites, small molecule substrates, intermediates and products of metabolism. The purpose of this study was to investigate the influence of sympathetic activation induced by a 1‐minute cold‐water immersion CPT, upon muscle metabolic responses, through metabolomics profiling of muscle interstitial fluid in adults. Five healthy adults (2 males, 3 females; 29.4 ± 9.4 yrs.; 77.6 ± 19.3 kg) were recruited for this pilot study. Three microdialysis probes (CMA 20, CMA/Microdialysis, Stockholm, Sweden) were percutaneously inserted into the vastus lateralis muscle of the dominant leg using sterile technique. Probes were then perfused with sterile saline containing 5 mM ethanol through the inlet tubing at a rate of 2.0 μL/min, and dialysate from the outlet tubing was collected in 150 μL polyethylene collection vials. One hour (3 × 20‐minute collections) of basal dialysates were collected and pooled. Participants then completed, in a seated position, a CPT in which the right hand was immersed up to the wrist in ice water for 1 minute. A 20‐min dialysate sample was collected during and immediately following the CPT. Microdialysis samples were sent to the West Coast Metabolomics Center (UC Davis Genome Center, Davis, CA, USA) for metabolomic profiling. Enzymatic‐fluorometric ethanol assay was also used to measure ethanol outflow: inflow (O:I) ratio to determine microvascular blood flow. The percent change from pre‐CPT to the CPT sample was calculated for every metabolite found. Data were then sorted on a magnitude basis, from smallest to largest change from basal levels. From the 413 metabolites detected, 207 were found to increase and 206 were found to decrease from baseline to post‐CPT. Nine metabolites were found within the 95th to 100th percentile of positive change from baseline to CPT, including: 1‐monostearin (median: 50.4%; range: 10.0–183.1), 3‐aminoisobutyric acid (median: 52.5%; range: 39.2–95.4), palmitoleic acid (median: 92.2%; range: 35.4–204.5); and 6 “unknown metabolites.” Furthermore, 5 “unknown metabolites” were also detected within the 95th to 100th percentile of negative change (decrease from baseline). Ethanol O:I ratio increased baseline to CPT, indicating decreased blood flow (0.483 ± 0.088 to 0.547 ± 0.027, mean ± SEM). These results indicate that the CPT can elicit both vasoconstriction and a notable metabolic response in muscle tissue, identifiable by the use of ethanol analysis and metabolomics of dialysate from microdialysis of skeletal muscle. Of specific interest, 1‐monostearin and palmitoleic acid were found to be in the group of molecules that increased the most in response to CPT. Although speculative, increased adrenergic stimulation resulting from CPT‐induced sympathetic activation may have resulted in liberation of 1‐monostearin and palmitoleic acid from membranes or other locations, or from increases in desaturase activities.

Biophysical Investigation of the Function of Enzyme 3DL1

Protein function is often tied to structure, so structural comparisons for a protein or enzyme of unknown function may help to elucidate its function. An enzyme of unknown function (PDB ID 3DL1) derived from Klebsiella pneumonia has been studied using various methods to predict and test enzyme function. Crystal structures of 3DL1 with and without zinc bound show that zinc binds at a site with sequence and structural similarity to known metallopeptidases. These crystal structures also show that zinc binding affects the secondary structure of the protein. Computational methods for sequence and structure comparisons were made of 3DL1 to other protein sequences and structures. These show that 3DL1 may be a hydrolase with possible peptidase activity. 3DL1 was expressed in E. coli cells and purified via metal-affinity chromatography. Size exclusion chromatography was used to further purify 3DL1. To study the secondary structure of 3DL1 in the presence and absence of zinc, circular dichroism (CD) spectroscopy was used. The CD data allowed for determination of the stoichiometry of binding of the zinc ion(s) to the protein. It was expected that one zinc ion would bind per protein molecule. Moreover, enzymatic assays were performed using various relevant substrates to monitor the activity of 3DL1 in the presence and absence of zinc. These assays were performed with small molecule substrates and were monitored by UV/visible spectroscopy. Because 3DL1 has some structural similarity to gelatinase A, the activity of 3DL1 with a larger protein substrate was also tested in a gelatin-based assay. The relevance of this project comes from the necessity to identify potential enzymatic action of proteins of unknown function. Through this, some proteins could become useful for treatment of human disease or lead to a greater understanding of their cause.

Combining free energy calculations with tailored enzyme activity assays to elucidate substrate binding of a phospho-lysine phosphatase.

Studying enzymes that are involved in the regulation of dynamic post-translational modifications (PTMs) is of key importance in proteomics research. Such investigations can be particularly challenging when the modification itself is intrinsically labile. In this article, we elucidate the enzymatic activity of Phospholysine Phosphohistidine Inorganic Pyrophosphate Phosphatase (LHPP) towards different O- and N-phosphorylated peptides by a combined experimental and computational approach. LHPP has been previously described to hydrolyze the phosphoramidate bonds in different small molecule substrates, including phosphorylated lysine (pLys). Taking the instability of the phosphoramidate bond into account, we conducted a carefully adjusted enzymatic assay with various pLys pentapeptides to confirm enzymatic phosphatase activity with LHPP. Molecular docking was employed to explore possible binding poses of the substrates in complex with the enzyme. Molecular dynamics based free energy calculations, which are unique in their accuracy and solid theoretical basis, were further applied to predict relative binding affinity of different substrates. Comparison of simulations with experiments clearly suggested a distinct binding motif of pLys peptides as well as a very narrow promiscuity of LHPP. We believe this integrated approach can be widely adopted to study the structure and interaction of poorly characterized enzyme–substrate complexes, in particular with synthetically challenging or labile substrates.

Facile addition of E-H bonds to a dicarbondiphosphide.

Charge transfer at P atoms in an N-heterocyclic carbene stabilized 6π-electron aromatic dicarbondiphosphide 1 has been observed upon interaction with a variety of small molecule substrates that feature a polar E-H bond (E = C, N, and O). As a result, these P atoms exhibit fleeting nucleophilicity and electrophilicity behaving as potential independent active sites, which effect the spontaneous addition of E-H bonds leading to a series of unsymmetrical 1,3-diphosphetane frameworks bearing P-C (2), P-N (6 and 7), and P-O bonds (8b, 8c, and 9). In addition, 1,3-diphosphetanium salts have been achieved when X-type substituents are weakly coordinating (i.e. [(CN)2CH]- (3), I- (4), and [(PhOH)2PhO]- (10)). The mechanisms of such transformations are investigated using density functional theory calculations.

Enzymatic promiscuity and the evolution of bioluminescence.

Bioluminescence occurs when an enzyme, known as a luciferase, oxidizes a small-molecule substrate, known as a luciferin. Nature has evolved multiple distinct luciferases and luciferins independently, all of which accomplish the impressive feat of light emission. One of the best-known examples of bioluminescence is exhibited by fireflies, a class of beetles that use d-luciferin as their substrate. The evolution of bioluminescence in beetles is thought to have emerged from ancestral fatty acyl-CoA synthetase (ACS) enzymes present in all insects. This theory is supported by multiple lines of evidence: Beetle luciferases share high sequence identity with these enzymes, often retain ACS activity, and some ACS enzymesfrom nonluminous insects can catalyze bioluminescence from synthetic d-luciferin analogues. Recent sequencing of firefly genomes and transcriptomes further illuminates how the duplication of ACS enzymesand subsequent diversification drove the evolution of bioluminescence. These genetic analyses have also uncovered candidate enzymes that may participate in luciferin metabolism. With the publication of the genomes and transcriptomes of fireflies and related insects, we are now better positioned to dissect and learn from the evolution of bioluminescence in beetles.

Rhodium-catalyzed reductive modification of pyrimidine nucleosides, nucleotide phosphates, and sugar nucleotides

Nucleosides and nucleotides are a group of small molecule effectors and substrates which include sugar nucleotides, purine and pyrimidine-based nucleotide phosphates, and diverse nucleotide antibiotics. We previously reported that hydrogenation of the nucleotide antibiotic tunicamycin leads to products with reduced toxicity on eukaryotic cells. We now report the hydrogenation of diverse sugar nucleosides, nucleotide phosphates, and pyrimidine nucleotides. UDP-sugars and other uridyl and thymidinyl nucleosides are quantitatively reduced to the corresponding 5,6-dihydro-nucleosides. Cytidyl pyrimidines are reduced, but the major products are the corresponding 5,6-dihydrouridyl nucleosides resulting from a deamination of the cytosine ring.

Biomimetic Mineralization of Protein Nanogels for Enzyme Protection.

Protein nanogels have found a wide variety of applications, ranging from biocatalysis to drug/protein delivery. However, in practical applications, proteins in nanogels may suffer from enzymic hydrolysis and denaturation. Inspired by the structure and functionalities of the fowl eggshells, biomimetic mineralization of protein nanogels was studied in this research. Protein nanogels with embedded porcine pancreas lipase (PPL) in the cross-linked nanostructures were synthesized through the thiol-disulfide reaction between thiol-functionalized PPL and poly(N-isopropylacrylamide) with pendant pyridyl disulfide groups. The nanogels were further reacted with reduced bovine serum albumin (BSA) and BSA molecules were coated on the nanogels. Mineralization of BSA leads to the synthesis of biomineralized shells on the nanogels. With the growth of CaCO3 on the shells, the nanogels aggregate into suprastructures. Thermogravimetric analysis, XRD, dynamic light scattering, and TEM were employed to study the mechanism of the biomineralization process and analyze the structures of the mineralized nanogels. The biomineralized shells can effectively protect the PPL molecules from hydrolysis by trypsin; meanwhile, the nanosized channels on the mineralized shells allow the transport of small-molecule substrates across the shells. Bioactivity measurements indicate that PPL in the nanogels maintains more than 80 % bioactivity after biomineralization.

Direct in Vitro Selection of Trans-Acting Ribozymes for Posttranscriptional, Site-Specific, and Covalent Fluorescent Labeling of RNA.

General and efficient tools for site-specific fluorescent or bioorthogonal labeling of RNA are in high demand. Here, we report direct in vitro selection, characterization, and application of versatile trans-acting 2'-5' adenylyl transferase ribozymes for covalent and site-specific RNA labeling. The design of our partially structured RNA pool allowed for in vitro evolution of ribozymes that modify a predetermined nucleotide in cis (i.e., intramolecular reaction) and can then be easily engineered for applications in trans (i.e., in an intermolecular setup). The resulting ribozymes are readily designed for specific target sites in small and large RNAs and accept a wide variety of N6-modified ATP analogues as small-molecule substrates. The most efficient new ribozyme (FH14) shows excellent specificity toward its target sequence also in the context of total cellular RNA.

Site-Selective C-H Halogenation Using Flavin-Dependent Halogenases Identified via Family-Wide Activity Profiling.

Enzymes are powerful catalysts for site-selective C–H bond functionalization. Identifying suitable enzymes for this task and for biocatalysis in general remains challenging, however, due to the fundamental difficulty of predicting catalytic activity from sequence information. In this study, family-wide activity profiling was used to obtain sequence–function information on flavin-dependent halogenases (FDHs). This broad survey provided a number of insights into FDH activity, including halide specificity and substrate preference, that were not apparent from the more focused studies reported to date. Regions of FDH sequence space that are most likely to contain enzymes suitable for halogenating small-molecule substrates were also identified. FDHs with novel substrate scope and complementary regioselectivity on large, three-dimensionally complex compounds were characterized and used for preparative-scale late-stage C–H functionalization. In many cases, these enzymes provide activities that required several rounds of directed evolution to accomplish in previous efforts, highlighting that this approach can achieve significant time savings for biocatalyst identification and provide advanced starting points for further evolution.

Planar three-coordinate iron sulfide in a synthetic [4Fe-3S] cluster with biomimetic reactivity.

Iron-sulfur clusters are emerging as reactive sites for the reduction of small-molecule substrates. However, the four-coordinate iron sites of typical iron-sulfur clusters rarely react with substrates, implicating three-coordinate iron. This idea is untested because fully sulfide-coordinated three-coordinate iron is unprecedented. Here we report a new type of [4Fe-3S] cluster featuring an iron center with three bonds to sulfides. Although a high-spin electronic configuration is characteristic of other iron-sulfur clusters, the planar geometry and short Fe–S bonds lead to a surprising low-spin electronic configuration at the three-coordinate Fe center as determined by spectroscopy and ab initio calculations. In a demonstration of biomimetic reactivity, the [4Fe-3S] cluster reduces hydrazine, a natural substrate of nitrogenase. The product is the first example of NH2 bound to an iron-sulfur cluster. Our results demonstrate that three-coordinate iron supported by sulfide donors is a plausible precursor to reactivity in iron-sulfur clusters like the FeMoco of nitrogenase.

Thermodynamics of H+/H•/H–/e– Transfer from [CpV(CO)3H]−: Comparisons to the Isoelectronic CpCr(CO)3H

Hydrogen atom (H•) donors generated from H2 facilitate the atom efficient reduction of small molecule substrates. However, generating H• donors with X–H bond dissociation free energies (BDFEs) belo...

Building Biological Flashlights: Orthogonal Luciferases and Luciferins for in Vivo Imaging.

Bioluminescence is widely used for real-time imaging in living organisms. This technology features a light-emitting reaction between enzymes (luciferases) and small molecule substrates (luciferins). Photons produced from luciferase-luciferin reactions can penetrate through heterogeneous tissue, enabling readouts of physiological processes. Dozens of bioluminescent probes are now available and many are routinely used to monitor cell proliferation, migration, and gene expression patterns in vivo. Despite the ubiquity of bioluminescence, traditional applications have been largely limited to imaging one biological feature at a time. Only a handful of luciferase-luciferin pairs can be easily used in tandem, and most are poorly resolved in living animals. Efforts to develop spectrally distinct reporters have been successful, but multispectral imaging in large organisms remains a formidable challenge due to interference from surrounding tissue. Consequently, a lack of well-resolved probes has precluded multicomponent tracking. An expanded collection of bioluminescent probes would provide insight into processes where multiple cell types drive physiological tasks, including immune function and organ development. We aimed to expand the bioluminescent toolkit by developing substrate-resolved imaging agents. The goal was to generate multiple orthogonal (i.e., noncross-reactive) luciferases that are responsive to unique scaffolds and could be used concurrently in living animals. We adopted a parallel engineering approach to genetically modify luciferases to accept chemically modified luciferins. When the mutants and analogs are combined, light is produced only when complementary enzyme-substrate partners interact. Thus, the pairs can be distinguished based on substrate selectivity, regardless of the color of light emitted. Sequential administration of the luciferins enables the unique luciferases to be illuminated (and thus resolved) within complex environments, including whole organisms. This Account describes our efforts to develop orthogonal bioluminescent probes, crafting custom luciferases (or "biological flashlights") that can selectively process luciferin analogs (or "batteries") to produce light. In the first section, we describe synthetic methods that were key to accessing diverse luciferin architectures. The second section focuses on identifying complementary luciferase enzymes via a combination of mutagenesis and screening. To expedite the search for orthogonal enzymes and substrates, we developed a computational algorithm to sift through large data sets. The third section features examples of the parallel engineering approach. We identified orthogonal enzyme-substrate pairs comprising two different classes of luciferins. The probes were vetted both in cells and whole organisms. This expanded collection of imaging agents is applicable to studies of immune function and other multicomponent processes. The final section of the Account highlights ongoing work toward building better bioluminescent tools. As ever-brighter and more selective probes are developed, the frontiers of what we can "see" in vivo will continue to expand.

Flexible Tethering of ASPP Proteins Facilitates PP-1c Catalysis

ASPP (apoptosis-stimulating proteins of p53) proteins bind PP-1c (protein phosphatase 1) and regulate p53 impacting cancer cell growth and apoptosis. Here we determine the crystal structure of the oncogenic ASPP protein, iASPP, bound to PP-1c. The structure reveals a 1:1 complex that relies on interactions of the iASPP SILK and RVxF motifs with PP-1c, plus interactions of the PP-1c PxxPxR motif with the iASPP SH3 domain. Small-angle X-ray scattering analyses suggest that the crystal structure undergoes slow interconversion with more extended conformations in solution. We show that iASPP, and the tumor suppressor ASPP2, enhance the catalytic activity of PP-1c against the small-molecule substrate, pNPP as well as p53. The combined results suggest that PxxPxR binding to iASPP SH3 domain is critical for complex formation, and that the modular ASPP-PP-1c interface provides dynamic flexibility that enables functional binding and dephosphorylation of p53 and other diverse protein substrates.

Graphite Conjugation Eliminates Redox Intermediates in Molecular Electrocatalysis.

The efficient interconversion of electrical and chemical energy requires the intimate coupling of electrons and small-molecule substrates at catalyst active sites. In molecular electrocatalysis, the molecule acts as a redox mediator which typically undergoes oxidation or reduction in a separate step from substrate activation. These mediated pathways introduce a high-energy intermediate, cap the driving force for substrate activation at the reduction potential of the molecule, and impede access to high rates at low overpotentials. Here we show that electronically coupling a molecular hydrogen evolution catalyst to a graphitic electrode eliminates stepwise pathways and forces concerted electron transfer and proton binding. Electrochemical and X-ray absorption spectroscopy data establish that hydrogen evolution catalysis at the graphite-conjugated Rh molecule proceeds without first reducing the metal center. These results have broad implications for the molecular-level design of energy conversion catalysts.

Inorganic Chemistry Approaches to Activity-Based Sensing: From Metal Sensors to Bioorthogonal Metal Chemistry.

The complex network of chemical processes that sustain life motivates the development of new synthetic tools to decipher biological mechanisms of action at a molecular level. In this context, fluorescent and related optical probes have emerged as useful chemical reagents for monitoring small-molecule and metal signals in biological systems, enabling visualization of dynamic cellular events with spatial and temporal resolution. In particular, metals occupy a central role in this field as analytes in their own right, while also being leveraged for their unique biocompatible reactivity with small-molecule substrates. This Viewpoint highlights the use of inorganic chemistry principles to develop activity-based sensing platforms mediated by metal reactivity, spanning indicators for metal detection to metal-based reagents for bioorthogonal tracking, and manipulation of small and large biomolecules, illustrating the privileged roles of metals at the interface of chemistry and biology.

Recent advances in the development of legumain-selective chemical probes and peptide prodrugs.

Legumain, which is also known as vacuolar processing enzyme (VPE) or asparaginyl endopeptidase (AEP), is a cysteine protease that was first discovered and characterized in the leguminous seeds of the moth bean in the early 1990s. Later, this enzyme was also detected in higher organisms, including eukaryotes. This pH-dependent protease displays the highest activity in acidic endolysosomal compartments; however, legumain also displays nuclear, cytosolic and extracellular activity when stabilized by other proteins or intramolecular complexes. Based on the results from over 25 years of research, this protease is involved in multiple cellular events, including protein degradation and antigen presentation. Moreover, when dysregulated, this protease contributes to the progression of several diseases, with cancer being the well-studied example. Research on legumain biology was undoubtedly facilitated by the use of small molecule chemical tools. Therefore, in this review, I present the historical perspectives and most current strategies for the development of small molecule substrates, inhibitors and activity-based probes for legumain. These tools are of paramount importance in elucidating the roles of legumain in multiple biological processes. Finally, as this enzyme appears to be a promising molecular target for anticancer therapies, the development of legumain-activated prodrugs is also described.