Mycofactocin-associated mycobacterial dehydrogenases with non-exchangeable NAD cofactors.

During human infection, Mycobacterium tuberculosis (Mtb) survives the normally bacteriocidal phagosome of macrophages. Mtb and related species may be able to combat this harsh acidic environment which contains reactive oxygen species due to the mycobacterial genomes encoding a large number of dehydrogenases. Typically, dehydrogenase cofactor binding sites are open to solvent, which allows NAD/NADH exchange to support multiple turnover. Interestingly, mycobacterial short chain dehydrogenases/reductases (SDRs) within family TIGR03971 contain an insertion at the NAD binding site. Here we present crystal structures of 9 mycobacterial SDRs in which the insertion buries the NAD cofactor except for a small portion of the nicotinamide ring. Line broadening and STD-NMR experiments did not show NAD or NADH exchange on the NMR timescale. STD-NMR demonstrated binding of the potential substrate carveol, the potential product carvone, the inhibitor tricyclazol, and an external redox partner 2,6-dichloroindophenol (DCIP). Therefore, these SDRs appear to contain a non-exchangeable NAD cofactor and may rely on an external redox partner, rather than cofactor exchange, for multiple turnover. Incidentally, these genes always appear in conjunction with the mftA gene, which encodes the short peptide MftA, and with other genes proposed to convert MftA into the external redox partner mycofactocin.

Synthesis and biological evaluation of pyrazolopyrimidines as potential antibacterial agents.

The fragment FOL7185 (compound 17) was found to be a hit against IspD and IspE enzymes isolated from bacteria, and a series of analogs containing the pyrazolopyrimidine core were synthesized. The majority of these compounds inhibited the growth of Burkholderia thailandensis (Bt) and Pseudomonas aeruginosa (Pa) in the Kirby­Bauer disk diffusion susceptibility test. Compound 29 shows inhibitory activity at 0.1 mM (32.2 lg/mL), which is comparable to the control compound kanamycin (48.5 lg/mL). Compound 29 also shows inhibitory activity at 0.5 mM against kanamycin resistant P. aeruginosa. Saturation transfer difference NMR (STD-NMR) screening of these compounds against BtIspD and BtIspE indicated that most of these compounds significantly interact with BtIspE, suggesting that the compounds may inhibit the growth of Bt by disrupting isoprenoid biosynthesis. Ligand epitope mapping of compound 29 with BtIspE indicated that hydrogens on 2,4-dichlorophenyl group have higher proximity to the surface of the enzyme than hydrogens on the pyrazolopyrimidine ring.

Increasing the structural coverage of tuberculosis drug targets.

High-resolution three-dimensional structures of essential Mycobacterium tuberculosis (Mtb) proteins provide templates for TB drug design, but are available for only a small fraction of the Mtb proteome. Here we evaluate an intra-genus "homolog-rescue" strategy to increase the structural information available for TB drug discovery by using mycobacterial homologs with conserved active sites. Of 179 potential TB drug targets selected for x-ray structure determination, only 16 yielded a crystal structure. By adding 1675 homologs from nine other mycobacterial species to the pipeline, structures representing an additional 52 otherwise intractable targets were solved. To determine whether these homolog structures would be useful surrogates in TB drug design, we compared the active sites of 106 pairs of Mtb and non-TB mycobacterial (NTM) enzyme homologs with experimentally determined structures, using three metrics of active site similarity, including superposition of continuous pharmacophoric property distributions. Pair-wise structural comparisons revealed that 19/22 pairs with >55% overall sequence identity had active site Cα RMSD <1 Å, >85% side chain identity, and ≥80% PSAPF (similarity based on pharmacophoric properties) indicating highly conserved active site shape and chemistry. Applying these results to the 52 NTM structures described above, 41 shared >55% sequence identity with the Mtb target, thus increasing the effective structural coverage of the 179 Mtb targets over three-fold (from 9% to 32%). The utility of these structures in TB drug design can be tested by designing inhibitors using the homolog structure and assaying the cognate Mtb enzyme; a promising test case, Mtb cytidylate kinase, is described. The homolog-rescue strategy evaluated here for TB is also generalizable to drug targets for other diseases.

Structural analysis of H1N1 and H7N9 influenza A virus PA in the absence of PB1.

Influenza A viruses cause the respiratory illness influenza, which can be mild to fatal depending on the strain and host immune response. The flu polymerase acidic (PA), polymerase basic 1 (PB1), and polymerase basic 2 (PB2) proteins comprise the RNA-dependent RNA polymerase complex responsible for viral genome replication. The first crystal structures of the C-terminal domain of PA (PA-CTD) in the absence of PB1-derived peptides show a number of structural changes relative to the previously reported PB1-peptide bound structures. The human A/WSN/1933 (H1N1) and avian A/Anhui1/2013 (H7N9) strain PA-CTD proteins exhibit the same global topology as other strains in the absence of PB1, but differ extensively in the PB1 binding pocket including a widening of the binding groove and the unfolding of a ß-turn. Both PA-CTD proteins exhibited a significant increase in thermal stability in the presence of either a PB1-derived peptide or a previously reported inhibitor in differential scanning fluorimetry assays. These structural changes demonstrate plasticity in the PA-PB1 binding interface which may be exploited in the development of novel therapeutics.

A structural biology approach enables the development of antimicrobials targeting bacterial immunophilins.

Macrophage infectivity potentiators (Mips) are immunophilin proteins and essential virulence factors for a range of pathogenic organisms. We applied a structural biology approach to characterize a Mip from Burkholderia pseudomallei (BpML1), the causative agent of melioidosis. Crystal structure and nuclear magnetic resonance analyses of BpML1 in complex with known macrocyclics and other derivatives led to the identification of a key chemical scaffold. This scaffold possesses inhibitory potency for BpML1 without the immunosuppressive components of related macrocyclic agents. Biophysical characterization of a compound series with this scaffold allowed binding site specificity in solution and potency determinations for rank ordering the set. The best compounds in this series possessed a low-micromolar affinity for BpML1, bound at the site of enzymatic activity, and inhibited a panel of homologous Mip proteins from other pathogenic bacteria, without demonstrating toxicity in human macrophages. Importantly, the in vitro activity of BpML1 was reduced by these compounds, leading to decreased macrophage infectivity and intracellular growth of Burkholderia pseudomallei. These compounds offer the potential for activity against a new class of antimicrobial targets and present the utility of a structure-based approach for novel antimicrobial drug discovery.

Cytidine derivatives as IspF inhibitors of Burkolderia pseudomallei.

Published biological data suggest that the methyl erythritol phosphate (MEP) pathway, a non-mevalonate isoprenoid biosynthetic pathway, is essential for certain bacteria and other infectious disease organisms. One highly conserved enzyme in the MEP pathway is 2C-methyl-d-erythritol 2,4-cyclodiphosphate synthase (IspF). Fragment-bound complexes of IspF from Burkholderia pseudomallei were used to design and synthesize a series of molecules linking the cytidine moiety to different zinc pocket fragment binders. Testing by surface plasmon resonance (SPR) found one molecule in the series to possess binding affinity equal to that of cytidine diphosphate, despite lacking any metal-coordinating phosphate groups. Close inspection of the SPR data suggest different binding stoichiometries between IspF and test compounds. Crystallographic analysis shows important variations between the binding mode of one synthesized compound and the pose of the bound fragment from which it was designed. The binding modes of these molecules add to our structural knowledge base for IspF and suggest future refinements in this compound series.

Multi-target parallel processing approach for gene-to-structure determination of the influenza polymerase PB2 subunit.

Pandemic outbreaks of highly virulent influenza strains can cause widespread morbidity and mortality in human populations worldwide. In the United States alone, an average of 41,400 deaths and 1.86 million hospitalizations are caused by influenza virus infection each year (1). Point mutations in the polymerase basic protein 2 subunit (PB2) have been linked to the adaptation of the viral infection in humans (2). Findings from such studies have revealed the biological significance of PB2 as a virulence factor, thus highlighting its potential as an antiviral drug target. The structural genomics program put forth by the National Institute of Allergy and Infectious Disease (NIAID) provides funding to Emerald Bio and three other Pacific Northwest institutions that together make up the Seattle Structural Genomics Center for Infectious Disease (SSGCID). The SSGCID is dedicated to providing the scientific community with three-dimensional protein structures of NIAID category A-C pathogens. Making such structural information available to the scientific community serves to accelerate structure-based drug design. Structure-based drug design plays an important role in drug development. Pursuing multiple targets in parallel greatly increases the chance of success for new lead discovery by targeting a pathway or an entire protein family. Emerald Bio has developed a high-throughput, multi-target parallel processing pipeline (MTPP) for gene-to-structure determination to support the consortium. Here we describe the protocols used to determine the structure of the PB2 subunit from four different influenza A strains.

Combining functional and structural genomics to sample the essential Burkholderia structome.

BACKGROUND: The genus Burkholderia includes pathogenic gram-negative bacteria that cause melioidosis, glanders, and pulmonary infections of patients with cancer and cystic fibrosis. Drug resistance has made development of new antimicrobials critical. Many approaches to discovering new antimicrobials, such as structure-based drug design and whole cell phenotypic screens followed by lead refinement, require high-resolution structures of proteins essential to the parasite. METHODOLOGY/PRINCIPAL FINDINGS: We experimentally identified 406 putative essential genes in B. thailandensis, a low-virulence species phylogenetically similar to B. pseudomallei, the causative agent of melioidosis, using saturation-level transposon mutagenesis and next-generation sequencing (Tn-seq). We selected 315 protein products of these genes based on structure-determination criteria, such as excluding very large and/or integral membrane proteins, and entered them into the Seattle Structural Genomics Center for Infection Disease (SSGCID) structure determination pipeline. To maximize structural coverage of these targets, we applied an "ortholog rescue" strategy for those producing insoluble or difficult to crystallize proteins, resulting in the addition of 387 orthologs (or paralogs) from seven other Burkholderia species into the SSGCID pipeline. This structural genomics approach yielded structures from 31 putative essential targets from B. thailandensis, and 25 orthologs from other Burkholderia species, yielding an overall structural coverage for 49 of the 406 essential gene families, with a total of 88 depositions into the Protein Data Bank. Of these, 25 proteins have properties of a potential antimicrobial drug target i.e., no close human homolog, part of an essential metabolic pathway, and a deep binding pocket. We describe the structures of several potential drug targets in detail. CONCLUSIONS/SIGNIFICANCE: This collection of structures, solubility and experimental essentiality data provides a resource for development of drugs against infections and diseases caused by Burkholderia. All expression clones and proteins created in this study are freely available by request.

Saturation transfer difference NMR for fragment screening.

Fragment screening by saturation transfer difference nuclear magnetic resonance (STD-NMR) is a robust method for identifying small molecule binders and is well suited to a broad set of biological targets. STD-NMR is exquisitely sensitive for detecting weakly binding compounds (a common characteristic of fragments), which is a crucial step in finding promising compounds for a fragment-based drug discovery campaign. This protocol describes the development of a library suitable for STD-NMR fragment screening, as well as preparation of protein samples, optimization of experimental conditions, and procedures for data collection and analysis.

Structural genomics of infectious disease drug targets: the SSGCID.

The Seattle Structural Genomics Center for Infectious Disease (SSGCID) is a consortium of researchers at Seattle BioMed, Emerald BioStructures, the University of Washington and Pacific Northwest National Laboratory that was established to apply structural genomics approaches to drug targets from infectious disease organisms. The SSGCID is currently funded over a five-year period by the National Institute of Allergy and Infectious Diseases (NIAID) to determine the three-dimensional structures of 400 proteins from a variety of Category A, B and C pathogens. Target selection engages the infectious disease research and drug-therapy communities to identify drug targets, essential enzymes, virulence factors and vaccine candidates of biomedical relevance to combat infectious diseases. The protein-expression systems, purified proteins, ligand screens and three-dimensional structures produced by SSGCID constitute a valuable resource for drug-discovery research, all of which is made freely available to the greater scientific community. This issue of Acta Crystallographica Section F, entirely devoted to the work of the SSGCID, covers the details of the high-throughput pipeline and presents a series of structures from a broad array of pathogenic organisms. Here, a background is provided on the structural genomics of infectious disease, the essential components of the SSGCID pipeline are discussed and a survey of progress to date is presented.

The Protein Maker: an automated system for high-throughput parallel purification.

The Protein Maker is an automated purification system developed by Emerald BioSystems for high-throughput parallel purification of proteins and antibodies. This instrument allows multiple load, wash and elution buffers to be used in parallel along independent lines for up to 24 individual samples. To demonstrate its utility, its use in the purification of five recombinant PB2 C-terminal domains from various subtypes of the influenza A virus is described. Three of these constructs crystallized and one diffracted X-rays to sufficient resolution for structure determination and deposition in the Protein Data Bank. Methods for screening lysis buffers for a cytochrome P450 from a pathogenic fungus prior to upscaling expression and purification are also described. The Protein Maker has become a valuable asset within the Seattle Structural Genomics Center for Infectious Disease (SSGCID) and hence is a potentially valuable tool for a variety of high-throughput protein-purification applications.

Probing conformational states of glutaryl-CoA dehydrogenase by fragment screening.

Glutaric acidemia type 1 is an inherited metabolic disorder which can cause macrocephaly, muscular rigidity, spastic paralysis and other progressive movement disorders in humans. The defects in glutaryl-CoA dehydrogenase (GCDH) associated with this disease are thought to increase holoenzyme instability and reduce cofactor binding. Here, the first structural analysis of a GCDH enzyme in the absence of the cofactor flavin adenine dinucleotide (FAD) is reported. The apo structure of GCDH from Burkholderia pseudomallei reveals a loss of secondary structure and increased disorder in the FAD-binding pocket relative to the ternary complex of the highly homologous human GCDH. After conducting a fragment-based screen, four small molecules were identified which bind to GCDH from B. pseudomallei. Complex structures were determined for these fragments, which cause backbone and side-chain perturbations to key active-site residues. Structural insights from this investigation highlight differences from apo GCDH and the utility of small-molecular fragments as chemical probes for capturing alternative conformational states of preformed protein crystals.

Inhibitor-bound complexes of dihydrofolate reductase-thymidylate synthase from Babesia bovis.

Babesiosis is a tick-borne disease caused by eukaryotic Babesia parasites which are morphologically similar to Plasmodium falciparum, the causative agent of malaria in humans. Like Plasmodium, different species of Babesia are tuned to infect different mammalian hosts, including rats, dogs, horses and cattle. Most species of Plasmodium and Babesia possess an essential bifunctional enzyme for nucleotide synthesis and folate metabolism: dihydrofolate reductase-thymidylate synthase. Although thymidylate synthase is highly conserved across organisms, the bifunctional form of this enzyme is relatively uncommon in nature. The structural characterization of dihydrofolate reductase-thymidylate synthase in Babesia bovis, the causative agent of babesiosis in livestock cattle, is reported here. The apo state is compared with structures that contain dUMP, NADP and two different antifolate inhibitors: pemetrexed and raltitrexed. The complexes reveal modes of binding similar to that seen in drug-resistant malaria strains and point to the utility of applying structural studies with proven cancer chemotherapies towards infectious disease research.

A small-molecule probe induces a conformation in HIV TAR RNA capable of binding drug-like fragments.

The HIV-1 transactivation response (TAR) element-Tat interaction is a potentially valuable target for treating HIV infection, but efforts to develop TAR-binding antiviral drugs have not yet yielded a successful candidate for clinical development. In this work, we describe a novel approach toward screening fragments against RNA that uses a chemical probe to target the Tat-binding region of TAR. This probe fulfills two critical roles in the screen: by locking the RNA into a conformation capable of binding other fragments, it simultaneously allows the identification of proximal binding fragments by ligand-based NMR. Using this approach, we have discovered six novel TAR-binding fragments, three of which were docked relative to the probe-RNA structure using experimental NMR restraints. The consistent orientations of functional groups in our data-driven docked structures and common electrostatic properties across all fragment leads reveal a surprising level of selectivity by our fragment-sized screening hits. These models further suggest linking strategies for the development of higher-affinity lead compounds for the inhibition of the TAR-Tat interaction.

Leveraging structure determination with fragment screening for infectious disease drug targets: MECP synthase from Burkholderia pseudomallei.

As part of the Seattle Structural Genomics Center for Infectious Disease, we seek to enhance structural genomics with ligand-bound structure data which can serve as a blueprint for structure-based drug design. We have adapted fragment-based screening methods to our structural genomics pipeline to generate multiple ligand-bound structures of high priority drug targets from pathogenic organisms. In this study, we report fragment screening methods and structure determination results for 2C-methyl-D-erythritol-2,4-cyclo-diphosphate (MECP) synthase from Burkholderia pseudomallei, the gram-negative bacterium which causes melioidosis. Screening by nuclear magnetic resonance spectroscopy as well as crystal soaking followed by X-ray diffraction led to the identification of several small molecules which bind this enzyme in a critical metabolic pathway. A series of complex structures obtained with screening hits reveal distinct binding pockets and a range of small molecules which form complexes with the target. Additional soaks with these compounds further demonstrate a subset of fragments to only bind the protein when present in specific combinations. This ensemble of fragment-bound complexes illuminates several characteristics of MECP synthase, including a previously unknown binding surface external to the catalytic active site. These ligand-bound structures now serve to guide medicinal chemists and structural biologists in rational design of novel inhibitors for this enzyme.

Predicting the success of fragment screening by X-ray crystallography.

Fragment screening using X-ray crystallography is a method that can provide direct three-dimensional readouts of the structures of protein-small molecule complexes for lead development and fragment-based drug discovery. With current technology, an amenable crystal form can be screened crystallographically against a library of 1000-2000 fragments in 1-2 weeks. We have performed over a dozen crystallographic screening campaigns using our own compound collection called Fragments of Life™ (FOL). While the majority of our fragment screening campaigns have generated multiple hits, some unexpectedly turned out to be nonproductive, either yielding no bound ligands, or only those thought to be inadequate for lead development. In this chapter, we have attempted to identify one or more parameters which could be used to predict whether a crystallized protein target would be a good candidate for fragment hit discovery. Here, we describe the parameters of crystals from 18 fragment screening campaigns, including six unsuccessful targets. From this analysis, we have concluded that there are no parameters that are absolutely predictive of fragment screening success. However, we do describe a parameter we have termed pocket factor which provides a statistically significant variance between nonproductive targets and productive targets shown to bind fragments. The pocket factor is calculated using a novel method of consensus scoring from three distinct pocket-finding algorithms, and the results may be used to prioritize targets for fragment screening campaigns based on an initial crystal structure.

Fragment screening of infectious disease targets in a structural genomics environment.

Structural genomics efforts have traditionally focused on generating single protein structures of unique and diverse targets. However, a lone structure for a given target is often insufficient to firmly assign function or to drive drug discovery. As part of the Seattle Structural Genomics Center for Infectious Disease (SSGCID), we seek to expand the focus of structural genomics by elucidating ensembles of structures that examine small molecule-protein interactions for selected infectious disease targets. In this chapter, we discuss two applications for small molecule libraries in structural genomics: unbiased fragment screening, to provide inspiration for lead development, and targeted, knowledge-based screening, to confirm or correct the functional annotation of a given gene product. This shift in emphasis results in a structural genomics effort that is more engaged with the infectious disease research community, and one that produces structures of greater utility to researchers interested in both protein function and inhibitor development. We also describe specific methods for conducting high-throughput fragment screening in a structural genomics context by X-ray crystallography.

Fragment-based discovery of novel thymidylate synthase leads by NMR screening and group epitope mapping.

Solution-state nuclear magnetic resonance (NMR) is a versatile tool for the study of binding interactions between small molecules and macromolecular targets. We applied ligand-based NMR techniques to the study of human thymidylate synthase (hTS) using known nanomolar inhibitors and a library of small molecule fragments. Screening by NMR led to the rapid identification of ligand pairs that bind in proximal sites within the cofactor-binding pocket of hTS. Screening hits were used as search criteria within commercially available sources, and a subset of catalog analogs were tested for potency by in vitro assay and binding affinity by quantitative saturation transfer difference (STD)-NMR titration. Two compounds identified by this approach possess low micromolar affinity and potency, as well as excellent binding efficiency against hTS. Relative binding orientations for both leads were modeled using AutoDock, and the most likely bound conformations were validated using experimentally derived STD-NMR binding epitope data. These ligands represent novel starting points for fragment-based drug design of non-canonical TS inhibitors, and their binding epitopes highlight important and previously unexploited interactions with conserved residues in the cofactor-binding site.