Synthesis and evaluation of dual-action kanglemycin-fluoroquinolone hybrid antibiotics.

Bacterial resistance threatens the utility of currently available antibiotics. Rifampicin, a cornerstone in the treatment of persistent Gram-positive infections, is prone to the development of resistance resulting from single point mutations in the antibiotic's target, RNA polymerase. One strategy to circumvent resistance is the use of 'hybrid' antibiotics consisting of two covalently linked antibiotic entities. These compounds generally have two distinct cellular targets, reducing the probability of resistance development and potentially providing simplified pharmacological properties compared to combination therapies using the individual antibiotics. Here we evaluate a series of semi-synthetic hybrid antibiotics formed by linking kanglemycin A (Kang A), a rifampicin analog, and a collection of fluoroquinolones. Kang A is a natural product antibiotic which contains a novel dimethyl succinic acid moiety that offers a new attachment point for the synthesis of hybrid antibiotics. We compare the activity of the Kang A hybrids generated via the acid attachment point to a series of hybrids linked at the compound's naphthoquinone ring system. Several hybrids exhibit activity against bacteria resistant to Kang A via the action of the partnered antibiotic, suggesting that the Kang scaffold may provide new avenues for generating antibiotics effective against drug-resistant infections.

A Semisynthetic Kanglemycin Shows In Vivo Efficacy against High-Burden Rifampicin Resistant Pathogens.

Semisynthetic rifamycin derivatives such as rifampicin (Rif) are first line treatments for tuberculosis and other bacterial infections. Historically, synthetic modifications made to the C-3/C-4 region of the rifamycin naphthalene core, like those seen in Rif, have yielded the biggest improvements in pharmacological properties. However, modifications found in natural product rifamycin congeners occur at other positions in the structure. The kanglemycins (Kangs) are a family of rifamycin congeners with a unique collection of natural modifications including a dimethylsuccinic acid appended to their polyketide backbone. These modifications confer activity against the single most common clinically relevant Rif resistance (RifR) mutation in the antibiotic's target, the bacterial RNA polymerase (RNAP). Here we evaluate the in vivo efficacy of Kang A, the parent compound in the Kang family, in a murine model of bacterial peritonitis/sepsis. We then set out to improve its potency by combining its natural tailoring modifications with semisynthetic derivatizations at either its acid moiety or in the C-3/C-4 region. A collection of C-3/C-4 benzoxazino Kang derivatives exhibit improved activity against wild-type bacteria, and acquire activity against the second most common clinically relevant RifR mutation. The semisynthetic analogue 3'-hydroxy-5'-[4-isobutyl-1-piperazinyl] benzoxazino Kang A (Kang KZ) protected mice against infection with either Rif sensitive MRSA or a highly virulent RifRStaphylococcus  aureus strain in a neutropenic peritonitis/sepsis model and led to reduced bacterial burdens. The compounds generated in this study may represent promising candidates for treating RifR infections.

Rifamycin congeners kanglemycins are active against rifampicin-resistant bacteria via a distinct mechanism.

Rifamycin antibiotics (Rifs) target bacterial RNA polymerases (RNAPs) and are widely used to treat infections including tuberculosis. The utility of these compounds is threatened by the increasing incidence of resistance (RifR). As resistance mechanisms found in clinical settings may also occur in natural environments, here we postulated that bacteria could have evolved to produce rifamycin congeners active against clinically relevant resistance phenotypes. We survey soil metagenomes and identify a tailoring enzyme-rich family of gene clusters encoding biosynthesis of rifamycin congeners (kanglemycins, Kangs) with potent in vivo and in vitro activity against the most common clinically relevant RifR mutations. Our structural and mechanistic analyses reveal the basis for Kang inhibition of RifR RNAP. Unlike Rifs, Kangs function through a mechanism that includes interfering with 5'-initiating substrate binding. Our results suggest that examining soil microbiomes for new analogues of clinically used antibiotics may uncover metabolites capable of circumventing clinically important resistance mechanisms.

Structural and biochemical approaches uncover multiple evolutionary trajectories of plant quinate dehydrogenases.

Quinate is produced and used by many plants in the biosynthesis of chlorogenic acids (CGAs). Chlorogenic acids are astringent and serve to deter herbivory. They also function as antifungal agents and have potent antioxidant properties. Quinate is produced at a branch point of shikimate biosynthesis by the enzyme quinate dehydrogenase (QDH). However, little information exists on the identity and biochemical properties of plant QDHs. In this study, we utilized structural and bioinformatics approaches to establish a QDH-specific primary sequence motif. Using this motif, we identified QDHs from diverse plants and confirmed their activity by recombinant protein production and kinetic assays. Through a detailed phylogenetic analysis, we show that plant QDHs arose directly from bifunctional dehydroquinate dehydratase-shikimate dehydrogenases (DHQD-SDHs) through different convergent evolutionary events, illustrated by our findings that eudicot and conifer QDHs arose early in vascular plant evolution whereas Brassicaceae QDHs emerged later. This process of recurrent evolution of QDH is further demonstrated by the fact that this family of proteins independently evolved NAD+ and NADP+ specificity in eudicots. The acquisition of QDH activity by these proteins was accompanied by the inactivation or functional evolution of the DHQD domain, as verified by enzyme activity assays and as reflected in the loss of key DHQD active site residues. The implications of QDH activity and evolution are discussed in terms of plant growth and development.

Structurally diverse dehydroshikimate dehydratase variants participate in microbial quinate catabolism.

Quinate and shikimate can be degraded by a number of microbes. Dehydroshikimate dehydratases (DSDs) play a central role in this process, catalyzing the conversion of 3-dehydroshikimate to protocatechuate, a common intermediate of aromatic degradation pathways. DSDs have applications in metabolic engineering for the production of valuable protocatechuate-derived molecules. Although a number of Gram-negative bacteria are known to catabolize quinate and shikimate, only limited information exists on the quinate/shikimate catabolic enzymes found in these organisms. Here, we have functionally and structurally characterized a putative DSD designated QuiC1, which is present in some pseudomonads. The QuiC1 protein is not related by sequence with previously identified DSDs from the Gram-negative genus, Acinetobacter, but instead shows limited sequence identity in its N-terminal half with fungal DSDs. Analysis of a Pseudomonas aeruginosa quiC1 gene knock-out demonstrates that it is important for growth on either quinate or shikimate. The structure of a QuiC1 enzyme from P. putida reveals that the protein is a fusion of two distinct modules: an N-terminal sugar phosphate isomerase-like domain associated with DSD activity and a novel C-terminal hydroxyphenylpyruvate dioxygenase-like domain. The results of this study highlight the considerable diversity of enzymes that participate in quinate/shikimate catabolism in different microbes.

The shikimate dehydrogenase family: functional diversity within a conserved structural and mechanistic framework.

Shikimate dehydrogenase (SDH) catalyzes the NADPH-dependent reduction of 3-deydroshikimate to shikimate, an essential reaction in the biosynthesis of the aromatic amino acids and a large number of other secondary metabolites in plants and microbes. The indispensible nature of this enzyme makes it a potential target for herbicides and antimicrobials. SDH is the archetypal member of a large protein family, which contains at least four additional functional classes with diverse metabolic roles. The different members of the SDH family share a highly similar three-dimensional structure and utilize a conserved catalytic mechanism, but exhibit distinct substrate preferences, making the family a particularly interesting system for studying modes of substrate recognition used by enzymes. Here, we review our current understanding of the biochemical and structural properties of each of the five previously identified SDH family functional classes.

Isolation and molecular characterization of the shikimate dehydrogenase domain from the Toxoplasma gondii AROM complex.

The apicomplexan parasite Toxoplasma gondii, the etiologic agent of toxoplasmosis, is estimated to infect 10-80% of different human populations. T. gondii encodes a large pentafunctional polypeptide known as the AROM complex which catalyzes five reactions in the shikimate pathway, a metabolic pathway required for the biosynthesis of the aromatic amino acids and a promising target for anti-parasitic agents. Here, we present the isolation, cloning and kinetic characterization of the shikimate dehydrogenase domain (TgSDH) from the T. gondii AROM complex. Recombinant TgSDH catalyzed the NADP(+)-dependent oxidation of shikimate in the absence of the remaining AROM domains and was sensitive to inhibition by a previously identified SDH inhibitor. Analysis of the TgSDH amino acid sequence revealed a number of novel insertions not found in SDH homologs from other organisms. Nevertheless, a three-dimensional structural model of TgSDH predicts a high level of conservation in the 'core' structure of the enzyme.

Identification of Novel Polyphenolic Inhibitors of Shikimate Dehydrogenase (AroE).

Shikimate dehydrogenase (AroE) is an attractive target for herbicides and antimicrobial agents due to its conserved and essential nature in plants, fungi, and bacteria. Here, we have performed an in vitro screen using a collection of more than 5500 compounds and identified 24 novel inhibitors of AroE from Pseudomonas putida The IC50 values for the two most potent inhibitors we identified, epigallocatechin gallate (EGCG) and epicatechin gallate (ECG), were 3.0 ± 0.2 µM and 3.7 ± 0.5 µM, respectively. Based on the high level of structural conservation between AroE orthologs, we predicted that the identified compounds would also inhibit AroE enzymes from other organisms. Consistent with this hypothesis, we found that EGCG and ECG inhibit the AroE domain of the bifunctional dehydroquinate dehydratase-shikimate dehydrogenase (DHQ-SDH) from Arabidopsis thaliana with IC50 values of 2.1 ± 0.3 µM and 2.0 ± 0.2 µM, respectively.

Insights into the function of RifI2: structural and biochemical investigation of a new shikimate dehydrogenase family protein.

The shikimate dehydrogenase (SDH) family consists of enzymes with diverse roles in secondary metabolism. The two most widespread members of the family, AroE and YdiB, function in amino acid biosynthesis and quinate catabolism, respectively. Here, we have determined the crystal structure of an SDH homolog belonging to the RifI class, a group of enzymes with proposed roles in antibiotic biosynthesis. The structure of RifI2 from Pseudomonas putida exhibits a number of distinctive features, including a substantial C-terminal truncation and an atypical mode of oligomerization. The active site of the enzyme contains substrate- and cofactor-binding motifs that are significantly different from those of any previously characterized member of the SDH family. These features are reflected in the novel kinetic properties of the enzyme. RifI2 exhibits much lower activity using shikimate as a substrate than AroE, and a strong preference for NAD(+) instead of NADP(+) as a cofactor. Moreover, the enzyme has only trace activity using quinate, unlike YdiB. Cocrystallization of RifI2 with NAD(+) provided the opportunity to determine the mode of cofactor selectivity employed by the enzyme. We complemented this analysis by probing the role of a strictly conserved residue in the cofactor-binding domain, Asn193, by site directed mutagenesis. This study presents the first crystal structure and formal kinetic characterization of a new NAD(+)-dependent member of the SDH family.

Structural and mechanistic analysis of a novel class of shikimate dehydrogenases: evidence for a conserved catalytic mechanism in the shikimate dehydrogenase family.

Shikimate dehydrogenase (SDH) catalyzes the reversible NADPH-dependent reduction of 3-dehydroshikimate to shikimate. This reaction represents the fourth step of the shikimate pathway, the essential route for the biosynthesis of the aromatic amino acids in plants, fungi, bacteria, and apicomplexan parasites. The absence of this pathway in animals makes it an attractive target for herbicides and antimicrobials. At least four functionally distinct enzyme classes, AroE, YdiB, SDH-like (SdhL), and AroE-like1 (Ael1), utilize shikimate as a substrate in vitro and form the SDH family. Crystal structures have been determined for AroE, YdiB, and SdhL. In this study, we have determined the first representative crystal structure of an Ael1 enzyme. We demonstrate that Ael1 shares a similar overall structure with the other members of the SDH family. This high level of structural conservation extends to the active sites of the enzymes. In particular, an ionizable active site lysine and aspartate are present in all SDH homologues. Two distinct biochemical roles have been reported for this Lys-Asp pair: as binding residues in YdiB and as a catalytic dyad in AroE and SdhL. Here, we establish that the residues function as a catalytic dyad in Ael1 and, interestingly, in at least one YdiB homologue. The conservation of three-dimensional fold, active site architecture, and catalytic mechanism among members of the SDH family will facilitate the design of drugs targeting the shikimate pathway.

A high-throughput chemical screen for resistance to Pseudomonas syringae in Arabidopsis.

The study of plant pathogenesis and the development of effective treatments to protect plants from diseases could be greatly facilitated by a high-throughput pathosystem to evaluate small-molecule libraries for inhibitors of pathogen virulence. The interaction between the Gram-negative bacterium Pseudomonas syringae and Arabidopsis thaliana is a model for plant pathogenesis. However, a robust high-throughput assay to score the outcome of this interaction is currently lacking. We demonstrate that Arabidopsis seedlings incubated with P. syringae in liquid culture display a macroscopically visible 'bleaching' symptom within 5 days of infection. Bleaching is associated with a loss of chlorophyll from cotyledonary tissues, and is correlated with bacterial virulence. Gene-for-gene resistance is absent in the liquid environment, possibly because of the suppression of the hypersensitive response under these conditions. Importantly, bleaching can be prevented by treating seedlings with known inducers of plant defence, such as salicylic acid (SA) or a basal defence-inducing peptide of bacterial flagellin (flg22) prior to inoculation. Based on these observations, we have devised a high-throughput liquid assay using standard 96-well plates to investigate the P. syringae-Arabidopsis interaction. An initial screen of small molecules active on Arabidopsis revealed a family of sulfanilamide compounds that afford protection against the bleaching symptom. The most active compound, sulfamethoxazole, also reduced in planta bacterial growth when applied to mature soil-grown plants. The whole-organism liquid assay provides a novel approach to probe chemical libraries in a high-throughput manner for compounds that reduce bacterial virulence in plants.