Evolutionary Genome Mining for the Discovery and Engineering of Natural Product Biosynthesis.

Genome mining has become an invaluable tool in natural products research to quickly identify and characterize the biosynthetic pathways that assemble secondary or specialized metabolites. Recently, evolutionary principles have been incorporated into genome mining strategies in an effort to better assess and prioritize novelty and understand their chemical diversification for engineering purposes. Here, we provide an introduction to the principles underlying evolutionary genome mining, including bioinformatic strategies and natural product biosynthetic databases. We introduce workflows for traditional genome mining, focusing on the popular pipeline antiSMASH, and methods to predict enzyme substrate specificity from genomic information. We then provide an in-depth discussion of evolutionary genome mining workflows, including EvoMining, CORASON, ARTS, and others, as adopted by our group for the discovery and prioritization of natural products biosynthetic gene clusters and their products.

Chemoselective ratiometric imaging of protein S-sulfenylation.

Here we report a ratiometric fluorescent probe for chemoselective conjugation to sulfenic acids in living cells. Our approach couples an α-fluoro-substituted dimedone to an aminonaphthalene fluorophore (F-DiNap), which upon sulfenic acid conjugation is locked as the 1,3-diketone, changing the fluorophore excitation. F-DiNap reacts with S-sulfenylated proteins at equivalent rates to current probes, but the α-fluorine substitution blocks side-reactions with biological aldehydes.

Molecular Mechanism for Isoform-Selective Inhibition of Acyl Protein Thioesterases 1 and 2 (APT1 and APT2).

Post-translational S-palmitoylation directs the trafficking and membrane localization of hundreds of cellular proteins, often involving a coordinated palmitoylation cycle that requires both protein acyl transferases (PATs) and acyl protein thioesterases (APTs) to actively redistribute S-palmitoylated proteins toward different cellular membrane compartments. This process is necessary for the trafficking and oncogenic signaling of S-palmitoylated Ras isoforms, and potentially many peripheral membrane proteins. The depalmitoylating enzymes APT1 and APT2 are separately conserved in all vertebrates, suggesting unique functional roles for each enzyme. The recent discovery of the APT isoform-selective inhibitors ML348 and ML349 has opened new possibilities to probe the function of each enzyme, yet it remains unclear how each inhibitor achieves orthogonal inhibition. Herein, we report the high-resolution structure of human APT2 in complex with ML349 (1.64 Å), as well as the complementary structure of human APT1 bound to ML348 (1.55 Å). Although the overall peptide backbone structures are nearly identical, each inhibitor adopts a distinct conformation within each active site. In APT1, the trifluoromethyl group of ML348 is positioned above the catalytic triad, but in APT2, the sulfonyl group of ML349 forms hydrogen bonds with active site resident waters to indirectly engage the catalytic triad and oxyanion hole. Reciprocal mutagenesis and activity profiling revealed several differing residues surrounding the active site that serve as critical gatekeepers for isoform accessibility and dynamics. Structural and biochemical analysis suggests the inhibitors occupy a putative acyl-binding region, establishing the mechanism for isoform-specific inhibition, hydrolysis of acyl substrates, and structural orthogonality important for future probe development.

Mechanisms of Resistance to Aminoglycoside Antibiotics: Overview and Perspectives.

Aminoglycoside (AG) antibiotics are used to treat many Gram-negative and some Gram-positive infections and, importantly, multidrug-resistant tuberculosis. Among various bacterial species, resistance to AGs arises through a variety of intrinsic and acquired mechanisms. The bacterial cell wall serves as a natural barrier for small molecules such as AGs and may be further fortified via acquired mutations. Efflux pumps work to expel AGs from bacterial cells, and modifications here too may cause further resistance to AGs. Mutations in the ribosomal target of AGs, while rare, also contribute to resistance. Of growing clinical prominence is resistance caused by ribosome methyltransferases. By far the most widespread mechanism of resistance to AGs is the inactivation of these antibiotics by AG-modifying enzymes. We provide here an overview of these mechanisms by which bacteria become resistant to AGs and discuss their prevalence and potential for clinical relevance.

Harnessing Redox Cross-Reactivity To Profile Distinct Cysteine Modifications.

Cysteine S-nitrosation and S-sulfination are naturally occurring post-translational modifications (PTMs) on proteins induced by physiological signals and redox stress. Here we demonstrate that sulfinic acids and nitrosothiols react to form a stable thiosulfonate bond, and leverage this reactivity using sulfinate-linked probes to enrich and annotate hundreds of endogenous S-nitrosated proteins. In physiological buffers, sulfinic acids do not react with iodoacetamide or disulfides, enabling selective alkylation of free thiols and site-specific analysis of S-nitrosation. In parallel, S-nitrosothiol-linked probes enable enrichment and detection of endogenous S-sulfinated proteins, confirming that a single sulfinic acid can react with a nitrosothiol to form a thiosulfonate linkage. Using this approach, we find that hydrogen peroxide addition increases S-sulfination of human DJ-1 (PARK7) at Cys106, whereas Cys46 and Cys53 are fully oxidized to sulfonic acids. Comparative gel-based analysis of different mouse tissues reveals distinct profiles for both S-nitrosation and S-sulfination. Quantitative proteomic analysis demonstrates that both S-nitrosation and S-sulfination are widespread, yet exhibit enhanced occupancy on select proteins, including thioredoxin, peroxiredoxins, and other validated redox active proteins. Overall, we present a direct, bidirectional method to profile select redox cysteine modifications based on the unique nucleophilicity of sulfinic acids.

Interrupted adenylation domains: unique bifunctional enzymes involved in nonribosomal peptide biosynthesis.

Nonribosomal peptides (NRPs) account for a large portion of drugs and drug leads currently available in the pharmaceutical industry. They are one of two main families of natural products biosynthesized on megaenzyme assembly-lines composed of multiple modules that are, in general, each comprised of three core domains and on occasion of accompanying auxiliary domains. The core adenylation (A) domains are known to delineate the identity of the specific chemical components to be incorporated into the growing NRPs. Previously believed to be inactive, A domains interrupted by auxiliary enzymes have recently been proven to be active and capable of performing two distinct chemical reactions. This highlight summarizes current knowledge on A domains and presents the various interrupted A domains found in a number of nonribosomal peptide synthetase (NRPS) assembly-lines, their predicted or proven dual functions, and their potential for manipulation and engineering for chemoenzymatic synthesis of new pharmaceutical agents with increased potency.

Enzymatic and cryoreduction EPR studies of the hydroxylation of methylated N(ω)-hydroxy-L-arginine analogues by nitric oxide synthase from Geobacillus stearothermophilus.

Nitric oxide synthase (NOS) catalyzes the conversion of L-arginine to L-citrulline and NO in a two-step process involving the intermediate N(ω)-hydroxy-L-arginine (NHA). It was shown that Cpd I is the oxygenating species for L-arginine; the hydroperoxo ferric intermediate is the reactive intermediate with NHA. Methylation of the N(ω)-OH and N(ω)-H of NHA significantly inhibits the conversion of NHA into NO and L-citrulline by mammalian NOS. Kinetic studies now show that N(ω)-methylation of NHA has a qualitatively similar effect on H2O2-dependent catalysis by bacterial gsNOS. To elucidate the effect of methylating N(ω)-hydroxy L-arginine on the properties and reactivity of the one-electron-reduced oxy-heme center of NOS, we have applied cryoreduction/annealing/EPR/ENDOR techniques. Measurements of solvent kinetic isotope effects during 160 K cryoannealing cryoreduced oxy-gsNOS/NHA confirm the hydroperoxo ferric intermediate as the catalytically active species of step two. Product analysis for cryoreduced samples with methylated NHA's, NHMA, NMOA, and NMMA, annealed to 273 K, show a correlation of yields of L-citrulline with the intensity of the g 2.26 EPR signal of the peroxo ferric species trapped at 77 K, which converts to the reactive hydroperoxo ferric state. There is also a correlation between the yield of L-citrulline in these experiments and k(obs) for the H2O2-dependent conversion of the substrates by gsNOS. Correspondingly, no detectable amount of cyanoornithine, formed when Cpd I is the reactive species, was found in the samples. Methylation of the NHA guanidinium N(ω)-OH and N(ω)-H inhibits the second NO-producing reaction by favoring protonation of the ferric-peroxo to form unreactive conformers of the ferric-hydroperoxo state. It is suggested that this is caused by modification of the distal-pocket hydrogen-bonding network of oxy gsNOS and introduction of an ordered water molecule that facilitates delivery of the proton(s) to the one-electron-reduced oxy-heme moiety. These results illustrate how variations in the properties of the substrate can modulate the reactivity of a monooxygenase.

Redesign of substrate specificity and identification of the aminoglycoside binding residues of Eis from Mycobacterium tuberculosis.

The upsurge in drug-resistant tuberculosis (TB) is an emerging global problem. The increased expression of the enhanced intracellular survival (Eis) protein is responsible for the clinical resistance to aminoglycoside (AG) antibiotics of Mycobacterium tuberculosis . Eis from M. tuberculosis (Eis_Mtb) and M. smegmatis (Eis_Msm) function as acetyltransferases capable of acetylating multiple amines of many AGs; however, these Eis homologues differ in AG substrate preference and in the number of acetylated amine groups per AG. The AG binding cavity of Eis_Mtb is divided into two narrow channels, whereas Eis_Msm contains one large cavity. Five bulky residues lining one of the AG binding channels of Eis_Mtb, His119, Ile268, Trp289, Gln291, and Glu401, have significantly smaller counterparts in Eis_Msm, Thr119, Gly266, Ala287, Ala289, and Gly401, respectively. To identify the residue(s) responsible for AG binding in Eis_Mtb and for the functional differences from Eis_Msm, we have generated single, double, triple, quadruple, and quintuple mutants of these residues in Eis_Mtb by mutating them into their Eis_Msm counterparts, and we tested their acetylation activity with three structurally diverse AGs: kanamycin A (KAN), paromomyin (PAR), and apramycin (APR). We show that penultimate C-terminal residue Glu401 plays a critical role in the overall activity of Eis_Mtb. We also demonstrate that the identities of residues Ile268, Trp289, and Gln291 (in Eis_Mtb nomenclature) dictate the differences between the acetylation efficiencies of Eis_Mtb and Eis_Msm for KAN and PAR. Finally, we show that the mutation of Trp289 in Eis_Mtb into Ala plays a role in APR acetylation.

Strategies to overcome the action of aminoglycoside-modifying enzymes for treating resistant bacterial infections.

Shortly after the discovery of the first antibiotics, bacterial resistance began to emerge. Many mechanisms give rise to resistance; the most prevalent mechanism of resistance to the aminoglycoside (AG) family of antibiotics is the action of aminoglycoside-modifying enzymes (AMEs). Since the identification of these modifying enzymes, many efforts have been put forth to prevent their damaging alterations of AGs. These diverse strategies are discussed within this review, including: creating new AGs that are unaffected by AMEs; developing inhibitors of AMEs to be co-delivered with AGs; or regulating AME expression. Modern high-throughput methods as well as drug combinations and repurposing are highlighted as recent drug-discovery efforts towards fighting the increasing antibiotic resistance crisis.

Synthesis and evaluation of hetero- and homodimers of ribosome-targeting antibiotics: antimicrobial activity, in vitro inhibition of translation, and drug resistance.

In this study, we describe the synthesis of a full set of homo- and heterodimers of three intact structures of different ribosome-targeting antibiotics: tobramycin, clindamycin, and chloramphenicol. Several aspects of the biological activity of the dimeric structures were evaluated including antimicrobial activity, inhibition of in vitro bacterial protein translation, and the effect of dimerization on the action of several bacterial resistance mechanisms that deactivate tobramycin and chloramphenicol. This study demonstrates that covalently linking two identical or different ribosome-targeting antibiotics may lead to (i) a broader spectrum of antimicrobial activity, (ii) improved inhibition of bacterial translation properties compared to that of the parent antibiotics, and (iii) reduction in the efficacy of some drug-modifying enzymes that confer high levels of resistance to the parent antibiotics from which the dimers were derived.

Methylated N(ω)-hydroxy-L-arginine analogues as mechanistic probes for the second step of the nitric oxide synthase-catalyzed reaction.

Nitric oxide synthase (NOS) catalyzes the conversion of L-arginine to L-citrulline through the intermediate N(ω)-hydroxy-L-arginine (NHA), producing nitric oxide, an important mammalian signaling molecule. Several disease states are associated with improper regulation of nitric oxide production, making NOS a therapeutic target. The first step of the NOS reaction has been well-characterized and is presumed to proceed through a compound I heme species, analogous to the cytochrome P450 mechanism. The second step, however, is enzymatically unprecedented and is thought to occur via a ferric peroxo heme species. To gain insight into the details of this unique second step, we report here the synthesis of NHA analogues bearing guanidinium methyl or ethyl substitutions and their investigation as either inhibitors of or alternate substrates for NOS. Radiolabeling studies reveal that N(ω)-methoxy-L-arginine, an alternative NOS substrate, produces citrulline, nitric oxide, and methanol. On the basis of these results, we propose a mechanism for the second step of NOS catalysis in which a methylated nitric oxide species is released and is further metabolized by NOS. Crystal structures of our NHA analogues bound to nNOS have been determined, revealing the presence of an active site water molecule only in the presence of singly methylated analogues. Bulkier analogues displace this active site water molecule; a different mechanism is proposed in the absence of the water molecule. Our results provide new insights into the steric and stereochemical tolerance of the NOS active site and substrate capabilities of NOS.

Target- and mechanism-based therapeutics for neurodegenerative diseases: strength in numbers.

The development of new therapeutics for the treatment of neurodegenerative pathophysiologies currently stands at a crossroads. This presents an opportunity to transition future drug discovery efforts to target disease modification, an area in which much still remains unknown. In this Perspective we examine recent progress in the areas of neurodegenerative drug discovery, focusing on some of the most common targets and mechanisms: N-methyl-d-aspartic acid (NMDA) receptors, voltage gated calcium channels (VGCCs), neuronal nitric oxide synthase (nNOS), oxidative stress from reactive oxygen species, and protein aggregation. These represent the key players identified in neurodegeneration and are part of a complex, intertwined signaling cascade. The synergistic delivery of two or more compounds directed against these targets, along with the design of small molecules with multiple modes of action, should be explored in pursuit of more effective clinical treatments for neurodegenerative diseases.

Cyclopropyl- and methyl-containing inhibitors of neuronal nitric oxide synthase.

Inhibitors of neuronal nitric oxide synthase have been proposed as therapeutics for the treatment of different types of neurological disorders. On the basis of a cis-3,4-pyrrolidine scaffold, a series of trans-cyclopropyl- and methyl-containing nNOS inhibitors have been synthesized. The insertion of a rigid electron-withdrawing cyclopropyl ring decreases the basicity of the adjacent amino group, which resulted in decreased inhibitory activity of these inhibitors compared to the parent compound. Nonetheless, three of them exhibited double-digit nanomolar inhibition with high nNOS selectivity on the basis of in vitro enzyme assays. Crystal structures of nNOS and eNOS with these inhibitors bound provide a basis for detailed structure-activity relationship (SAR) studies. The conclusions from these studies will be used as a guide in the future development of selective NOS inhibitors.

Selective monocationic inhibitors of neuronal nitric oxide synthase. Binding mode insights from molecular dynamics simulations.

The reduction of pathophysiologic levels of nitric oxide through inhibition of neuronal nitric oxide synthase (nNOS) has the potential to be therapeutically beneficial in various neurodegenerative diseases. We have developed a series of pyrrolidine-based nNOS inhibitors that exhibit excellent potencies and isoform selectivities (J. Am. Chem. Soc. 2010, 132, 5437). However, there are still important challenges, such as how to decrease the multiple positive charges derived from basic amino groups, which contribute to poor bioavailability, without losing potency and/or selectivity. Here we present an interdisciplinary study combining molecular docking, crystallography, molecular dynamics simulations, synthesis, and enzymology to explore potential pharmacophoric features of nNOS inhibitors and to design potent and selective monocationic nNOS inhibitors. The simulation results indicate that different hydrogen bond patterns, electrostatic interactions, hydrophobic interactions, and a water molecule bridge are key factors for stabilizing ligands and controlling ligand orientation. We find that a heteroatom in the aromatic head or linker chain of the ligand provides additional stability and blocks the substrate binding pocket. Finally, the computational insights are experimentally validated with double-headed pyridine analogues. The compounds reported here are among the most potent and selective monocationic pyrrolidine-based nNOS inhibitors reported to date, and 10 shows improved membrane permeability.

Probing the Arabidopsis flagellin receptor: FLS2-FLS2 association and the contributions of specific domains to signaling function.

Flagellin sensing2 (FLS2) is a transmembrane receptor kinase that activates antimicrobial defense responses upon binding of bacterial flagellin or the flagellin-derived peptide flg22. We find that some Arabidopsis thaliana FLS2 is present in FLS2-FLS2 complexes before and after plant exposure to flg22. flg22 binding capability is not required for FLS2-FLS2 association. Cys pairs flank the extracellular leucine rich repeat (LRR) domain in FLS2 and many other LRR receptors, and we find that the Cys pair N-terminal to the FLS2 LRR is required for normal processing, stability, and function, possibly due to undescribed endoplasmic reticulum quality control mechanisms. By contrast, disruption of the membrane-proximal Cys pair does not block FLS2 function, instead increasing responsiveness to flg22, as indicated by a stronger oxidative burst. There was no evidence for intermolecular FLS2-FLS2 disulfide bridges. Truncated FLS2 containing only the intracellular domain associates with full-length FLS2 and exerts a dominant-negative effect on wild-type FLS2 function that is dependent on expression level but independent of the protein kinase capacity of the truncated protein. FLS2 is insensitive to disruption of multiple N-glycosylation sites, in contrast with the related receptor EF-Tu receptor that can be rendered nonfunctional by disruption of single glycosylation sites. These and additional findings more precisely define the molecular mechanisms of FLS2 receptor function.

Intramolecular hydrogen bonding: a potential strategy for more bioavailable inhibitors of neuronal nitric oxide synthase.

Selective neuronal nitric oxide synthase (nNOS) inhibitors have therapeutic applications in the treatment of numerous neurodegenerative diseases. Here we report the synthesis and evaluation of a series of inhibitors designed to have increased cell membrane permeability via intramolecular hydrogen bonding. Their potencies were examined in both purified enzyme and cell-based assays; a comparison of these results demonstrates that two of the new inhibitors display significantly increased membrane permeability over previous analogs. NMR spectroscopy provides evidence of intramolecular hydrogen bonding under physiological conditions in two of the inhibitors. Crystal structures of the inhibitors in the nNOS active site confirm the predicted non-intramolecular hydrogen bonded binding mode. Intramolecular hydrogen bonding may be an effective approach for increasing cell membrane permeability without affecting target protein binding.

Improved synthesis of chiral pyrrolidine inhibitors and their binding properties to neuronal nitric oxide synthase.

We report an efficient synthetic route to chiral pyrrolidine inhibitors of neuronal nitric oxide synthase (nNOS) and crystal structures of the inhibitors bound to nNOS and to endothelial NOS. The new route enables versatile structure-activity relationship studies on the pyrrolidine-based scaffold, which can be beneficial for further development of nNOS inhibitors. The X-ray crystal structures of five new fluorine-containing inhibitors bound to nNOS provide insights into the effect of the fluorine atoms on binding.