Samuel H. Light

Identification of novel small molecule inhibitors against NS2B/NS3 serine protease from Zika virus

&NA; Zika flavivirus infection during pregnancy appears to produce higher risk of microcephaly, and also causes multiple neurological problems such as Guillain–Barré syndrome. The Zika virus is now widespread in Central and South America, and is anticipated to become an increasing risk in the southern United States. With continuing global travel and the spread of the mosquito vector, the exposure is expected to accelerate, but there are no currently approved treatments against the Zika virus. The Zika NS2B/NS3 protease is an attractive drug target due to its essential role in viral replication. Our studies have identified several compounds with inhibitory activity (IC50) and binding affinity (KD) of ˜5–10 &mgr;M against the Zika NS2B‐NS3 protease from testing 71 HCV NS3/NS4A inhibitors that were initially discovered by high‐throughput screening of 40,967 compounds. Competition surface plasmon resonance studies and mechanism of inhibition analyses by enzyme kinetics subsequently determined the best compound to be a competitive inhibitor with a Ki value of 9.5 &mgr;M. We also determined the X‐ray structure of the Zika NS2B‐NS3 protease in a “pre‐open conformation”, a conformation never observed before for any flavivirus proteases. This provides the foundation for new structure‐based inhibitor design. Graphical abstract Figure. No caption available. HighlightsWe identified several small molecule inhibitors of the Zika NS2B‐NS3 protease.Two lead compounds were determined to be competitive inhibitors by both enzymatic and competition SPR binding analyses.We determined the X‐ray structure of the apo Zika NS2B‐NS3 protease in a “pre‐open conformation”.

Insights into the mechanism of type I dehydroquinate dehydratases from structures of reaction intermediates.

The biosynthetic shikimate pathway consists of seven enzymes that catalyze sequential reactions to generate chorismate, a critical branch point in the synthesis of the aromatic amino acids. The third enzyme in the pathway, dehydroquinate dehydratase (DHQD), catalyzes the dehydration of 3-dehydroquinate to 3-dehydroshikimate. We present three crystal structures of the type I DHQD from the intestinal pathogens Clostridium difficile and Salmonella enterica. Structures of the enzyme with substrate and covalent pre- and post-dehydration reaction intermediates provide snapshots of successive steps along the type I DHQD-catalyzed reaction coordinate. These structures reveal that the position of the substrate within the active site does not appreciably change upon Schiff base formation. The intermediate state structures reveal a reaction state-dependent behavior of His-143 in which the residue adopts a conformation proximal to the site of catalytic dehydration only when the leaving group is present. We speculate that His-143 is likely to assume differing catalytic roles in each of its observed conformations. One conformation of His-143 positions the residue for the formation/hydrolysis of the covalent Schiff base intermediates, whereas the other conformation positions the residue for a role in the catalytic dehydration event. The fact that the shikimate pathway is absent from humans makes the enzymes of the pathway potential targets for the development of non-toxic antimicrobials. The structures and mechanistic insight presented here may inform the design of type I DHQD enzyme inhibitors.

The diversity of allosteric controls at the gateway to aromatic amino acid biosynthesis

Present within bacteria, plants, and some lower eukaryotes 3‐deoxy‐D‐arabino‐heptulosonate 7‐phosphate synthase (DAHPS) catalyzes the first committed step in the synthesis of a number of metabolites, including the three aromatic amino acids phenylalanine, tyrosine, and tryptophan. Catalyzing the first reaction in an important biosynthetic pathway, DAHPS is situated at a critical regulatory checkpoint—at which pathway input can be efficiently modulated to respond to changes in the concentration of pathway outputs. Based on a phylogenetic classification scheme, DAHPSs have been divided into three major subtypes (Iα, Iβ, and II). These subtypes are subjected to an unusually diverse pattern of allosteric regulation, which can be used to further subdivide the enzymes. Crystal structures of most of the regulatory subclasses have been determined. When viewed collectively, these structures illustrate how distinct mechanisms of allostery are applied to a common catalytic scaffold. Here, we review structural revelations regarding DAHPS regulation and make the case that the functional difference between the three major DAHPS subtypes relates to basic distinctions in quaternary structure and mechanism of allostery.

Structural analysis of a 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase with an N-terminal chorismate mutase-like regulatory domain

3‐Deoxy‐D‐arabino‐heptulosonate 7‐phosphate synthase (DAHPS) catalyzes the first step in the biosynthesis of a number of aromatic metabolites. Likely because this reaction is situated at a pivotal biosynthetic gateway, several DAHPS classes distinguished by distinct mechanisms of allosteric regulation have independently evolved. One class of DAHPSs contains a regulatory domain with sequence homology to chorismate mutase—an enzyme further downstream of DAHPS that catalyzes the first committed step in tyrosine/phenylalanine biosynthesis—and is inhibited by chorismate mutase substrate (chorismate) and product (prephenate). Described in this work, structures of the Listeria monocytogenes chorismate/prephenate regulated DAHPS in complex with Mn2+ and Mn2+ + phosphoenolpyruvate reveal an unusual quaternary architecture: DAHPS domains assemble as a tetramer, from either side of which chorismate mutase‐like (CML) regulatory domains asymmetrically emerge to form a pair of dimers. This domain organization suggests that chorismate/prephenate binding promotes a stable interaction between the discrete regulatory and catalytic domains and supports a mechanism of allosteric inhibition similar to tyrosine/phenylalanine control of a related DAHPS class. We argue that the structural similarity of chorismate mutase enzyme and CML regulatory domain provides a unique opportunity for the design of a multitarget antibacterial.

A conserved surface loop in type I dehydroquinate dehydratases positions an active site arginine and functions in substrate binding.

Dehydroquinate dehydratase (DHQD) catalyzes the third step in the biosynthetic shikimate pathway. We present three crystal structures of the Salmonella enterica type I DHQD that address the functionality of a surface loop that is observed to close over the active site following substrate binding. Two wild-type structures with differing loop conformations and kinetic and structural studies of a mutant provide evidence of both direct and indirect mechanisms of involvement of the loop in substrate binding. In addition to allowing amino acid side chains to establish a direct interaction with the substrate, closure of the loop necessitates a conformational change of a key active site arginine, which in turn positions the substrate productively. The absence of DHQD in humans and its essentiality in many pathogenic bacteria make the enzyme a target for the development of nontoxic antimicrobials. The structures and ligand binding insights presented here may inform the design of novel type I DHQD inhibiting molecules.

The structure of bradyzoite-specific enolase from Toxoplasma gondii reveals insights into its dual cytoplasmic and nuclear functions

The second crystal structure of a parasite protein preferentially enriched in the brain cyst of T. gondii has been solved at 2.75 Å resolution. Bradyzoite enolase 1 is reported to have differential functions as a glycolytic enzyme and a transcriptional regulator in bradyzoites.

Discovery of selective inhibitors of the Clostridium difficile dehydroquinate dehydratase.

A vibrant and healthy gut flora is essential for preventing the proliferation of Clostridium difficile, a pathogenic bacterium that causes severe gastrointestinal symptoms. In fact, most C. difficile infections (CDIs) occur after broad-spectrum antibiotic treatment, which, by eradicating the commensal gut bacteria, allows its spores to proliferate. Hence, a C. difficile specific antibiotic that spares the gut flora would be highly beneficial in treating CDI. Towards this goal, we set out to discover small molecule inhibitors of the C. difficile enzyme dehydroquinate dehydratase (DHQD). DHQD is the 3rd of seven enzymes that compose the shikimate pathway, a metabolic pathway absent in humans, and is present in bacteria as two phylogenetically and mechanistically distinct types. Using a high-throughput screen we identified three compounds that inhibited the type I C. difficile DHQD but not the type II DHQD from Bacteroides thetaiotaomicron, a highly represented commensal gut bacterial species. Kinetic analysis revealed that the compounds inhibit the C. difficile enzyme with Ki values ranging from 10 to 20 µM. Unexpectedly, kinetic and biophysical studies demonstrate that inhibitors also exhibit selectivity between type I DHQDs, inhibiting the C. difficile but not the highly homologous Salmonella enterica DHQD. Therefore, the three identified compounds seem to be promising lead compounds for the development of C. difficile specific antibiotics.

Adherence to Burgi-Dunitz stereochemical principles requires significant structural rearrangements in Schiff-base formation: insights from transaldolase complexes.

The Bürgi-Dunitz angle (αBD) describes the trajectory of approach of a nucleophile to an electrophile. The adoption of a stereoelectronically favorable αBD can necessitate significant reactive-group repositioning over the course of bond formation. In the context of enzyme catalysis, interactions with the protein constrain substrate rotation, which could necessitate structural transformations during bond formation. To probe this theoretical framework vis-à-vis biocatalysis, Schiff-base formation was analysed in Francisella tularensis transaldolase (TAL). Crystal structures of wild-type and Lys→Met mutant TAL in covalent and noncovalent complexes with fructose 6-phosphate and sedoheptulose 7-phosphate clarify the mechanism of catalysis and reveal that substrate keto moieties undergo significant conformational changes during Schiff-base formation. Structural changes compelled by the trajectory considerations discussed here bear relevance to bond formation in a variety of constrained enzymic/engineered systems and can inform the design of covalent therapeutics.

Crystal structures of type I dehydroquinate dehydratase in complex with quinate and shikimate suggest a novel mechanism of Schiff base formation.

A component of the shikimate biosynthetic pathway, dehydroquinate dehydratase (DHQD) catalyzes the dehydration of 3-dehydroquniate (DHQ) to 3-dehydroshikimate. In the type I DHQD reaction mechanism a lysine forms a Schiff base intermediate with DHQ. The Schiff base acts as an electron sink to facilitate the catalytic dehydration. To address the mechanism of Schiff base formation, we determined structures of the Salmonella enterica wild-type DHQD in complex with the substrate analogue quinate and the product analogue shikimate. In addition, we determined the structure of the K170M mutant (Lys170 being the Schiff base forming residue) in complex with quinate. Combined with nuclear magnetic resonance and isothermal titration calorimetry data that revealed altered binding of the analogue to the K170M mutant, these structures suggest a model of Schiff base formation characterized by the dynamic interplay of opposing forces acting on either side of the substrate. On the side distant from the substrate 3-carbonyl group, closure of the enzyme’s β8−α8 loop is proposed to guide DHQ into the proximity of the Schiff base-forming Lys170. On the 3-carbonyl side of the substrate, Lys170 sterically alters the position of DHQ’s reactive ketone, aligning it at an angle conducive for nucleophilic attack. This study of a type I DHQD reveals the interplay between the enzyme and substrate required for the correct orientation of a functional group constrained within a cyclic substrate.

Arabinose 5-phosphate covalently inhibits transaldolase

Arabinose 5-phosphate (A5P) is the aldopentose version of the ketohexose fructose 6-phosphate (F6P), having identical stereochemistry but lacking atoms corresponding to the 1-carbon and 1-hydroxyl. Despite structural similarity and conservation of the reactive portion of F6P, F6P acts as a substrate whereas A5P is reported to be an inhibitor of transaldolase. To address the lack of A5P reactivity we determined a crystal structure of the Francisella tularensis transaldolase in complex with A5P. This structure reveals that like F6P, A5P forms a covalent Schiff base with active site Lys135. Unlike F6P, A5P binding fails to displace an ordered active site water molecule. Retaining this water necessitates conformational changes at the A5P-protein linkage that possibly hinder reactivity. The findings presented here show the basis of A5P inhibition and suggest an unusual mechanism of competitive, reversible-covalent transaldolase regulation.