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Rationale High-throughput screening (HTS) is a critical step in the drug discovery process. However, most mass spectrometry-based HTS methods require sample cleanup steps prior to analysis. In this work we present the utility of infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) for monitoring an enzymatic reaction directly from a biological buffer system with no sample cleanup and at high throughput. Methods IR-MALDESI was used to directly analyze reaction mixtures from a well plate at different time points after reaction initiation. The percent conversion of precursors to products was used to screen the enzyme activity. The reaction was performed with two different concentrations of precursors and enzyme in order to assess the dynamic range of the assay. Eventually, a pseudo-HTS study was designed to investigate the utility of IR-MALDESI screening enzyme activity in a high-throughput manner. Results IR-MALDESI was able to readily monitor the activity of IDH1 over time at two different concentrations of precursors and enzyme. The calculated Z-factors of 0.65 and 0.41 confirmed the suitability of the developed method for screening enzyme activity in HTS manner. Finally, in a single-blind pseudo-HTS analysis IR-MALDESI was able to correctly predict the identity of all samples, where 8/10 samples were identified with high confidence and the other 2 samples with lower confidence. Conclusions The enzymatic activity of IDH1 was screened by directly analyzing the reaction content from the buffer in well plates with no sample cleanup steps. This proof-of-concept study demonstrates the robustness of IR-MALDESI for direct analysis of enzymatic reactions from biological buffers with no sample cleanup and its immense potential for HTS applications.

The primary objective of early drug discovery is to associate druggable target space with a desired phenotype. The inability to efficiently associate these often leads to failure early in the drug discovery process. In this proof-of-concept study, the most tractable starting points for drug discovery within the NF-κB pathway model system were identified by integrating affinity selection-mass spectrometry (AS-MS) with functional cellular assays. The AS-MS platform Automated Ligand Identification System (ALIS) was used to rapidly screen 15 NF-κB proteins in parallel against large-compound libraries. ALIS identified 382 target-selective compounds binding to 14 of the 15 proteins. Without any chemical optimization, 22 of the 382 target-selective compounds exhibited a cellular phenotype consistent with the respective target associated in ALIS. Further studies on structurally related compounds distinguished two chemical series that exhibited a preliminary structure-activity relationship and confirmed target-driven cellular activity to NF-κB1/p105 and TRAF5, respectively. These two series represent new drug discovery opportunities for chemical optimization. The results described herein demonstrate the power of combining ALIS with cell functional assays in a high-throughput, target-based approach to determine the most tractable drug discovery opportunities within a pathway.

Crystal structures of toluene 4-monooxygenase hydroxylase in complex with reaction products and effector protein reveal active site interactions leading to regiospecificity. Complexes with phenolic products yield an asymmetric μ-phenoxo-bridged diiron center and a shift of diiron ligand E231 into a hydrogen bonding position with conserved T201. In contrast, complexes with inhibitors p-NH(2)-benzoate and p-Br-benzoate showed a μ-1,1 coordination of carboxylate oxygen between the iron atoms and only a partial shift in the position of E231. Among active site residues, F176 trapped the aromatic ring of products against a surface of the active site cavity formed by G103, E104 and A107, while F196 positioned the aromatic ring against this surface via a π-stacking interaction. The proximity of G103 and F176 to the para substituent of the substrate aromatic ring and the structure of G103L T4moHD suggest how changes in regiospecificity arise from mutations at G103. Although effector protein binding produced significant shifts in the positions of residues along the outer portion of the active site (T201, N202, and Q228) and in some iron ligands (E231 and E197), surprisingly minor shifts (

RORγt is critical for the differentiation and proliferation of Th17 cells associated with several chronic autoimmune diseases. We report the discovery of a novel allosteric binding site on the nuclear receptor RORγt. Co-crystallization of the ligand binding domain (LBD) of RORγt with a series of small-molecule antagonists demonstrates occupancy of a previously unreported allosteric binding pocket. Binding at this non-canonical site induces an unprecedented conformational reorientation of helix 12 in the RORγt LBD, which blocks cofactor binding. The functional consequence of this allosteric ligand-mediated conformation is inhibition of function as evidenced by both biochemical and cellular studies. RORγt function is thus antagonized in a manner molecularly distinct from that of previously described orthosteric RORγt ligands. This brings forward an approach to target RORγt for the treatment of Th17-mediated autoimmune diseases. The elucidation of an unprecedented modality of pharmacological antagonism establishes a mechanism for modulation of nuclear receptors., Upon the binding of small ligands, nuclear receptors regulate the transcription of genes that are associated with a number of disease mechanisms. Here, the authors report on a novel allosteric ligand binding site on the nuclear receptor RORγt.

Clostridium thermocellum is a cellulosome-producing bacterium that is able to efficiently degrade and utilize cellulose as a sole carbon source. Cellobiose phosphorylase (CBP) plays a critical role in cellulose degradation by catalyzing the reversible phosphate-dependent hydrolysis of cellobiose, the major product of cellulose degradation, into α-D-glucose 1-phosphate and D-glucose. CBP from C. thermocellum is a modular enzyme composed of four domains [N-terminal domain, helical linker, (α/α)(6)-barrel domain and C-terminal domain] and is a member of glycoside hydrolase family 94. The 2.4 Å resolution X-ray crystal structure of C. thermocellum CBP reveals the residues involved in coordinating the catalytic phosphate as well as the residues that are likely to be involved in substrate binding and discrimination.

Toluene 4-monooxygenase (T4MO) is a member of the bacterial multicomponent monooxygenases, an enzyme family that utilizes a soluble diiron hydroxylase to oxidize a variety of hydrocarbons as the initial step in their metabolism. The hydroxylases obtain reducing equivalents from NAD(P)H via an electron transfer chain that is initiated by an oxidoreductase containing an N-terminal ferredoxin domain and C-terminal flavin- and NAD-binding domains. T4moF, the NADH oxidoreductase of T4MO, was expressed as a soluble protein in Escherichia coli BL21(DE3) from the pUC-derived expression vector pRS205. This vector contains a lac promoter instead of a T7 promoter. A three step purification from the soluble cell lysate yielded approximately 1 mg of T4moF per gram of wet cell paste with greater than 90% purity. The purified protein contained 1 mol of FAD and 2 mol of Fe per mol of T4moF; quantitative EPR spectroscopy showed approximately 1 mol of the S=1/2 signal from the reduced [2Fe-2S] cluster per mol of T4moF. Steady state kinetic analysis of p-cresol formation activity treating T4moF as the variable substrate while all other proteins and substrates were held constant gave apparent K(M-) and apparent k(cat)-values of 0.15 microM and 3.0 s(-1), respectively. This expression system and purification allows for the recovery of the soluble oxidoreductase in yields that facilitate further biochemical and structural characterizations.

Toluene 4-monooxygenase catalyzes the NADH- and O2-dependent hydroxylation of toluene to form p-cresol. The four-protein complex consists of a diiron hydroxylase, an oxidoreductase, a catalytic effector protein, and a Rieske-type ferredoxin (T4moC). Phylogenetic analysis suggests that T4moC is part of a clade specialized for reaction with diiron hydroxylases, possibly reflected in the conservation of W69, whose indole side chain makes close contacts with a bridging sulfide. In order to further investigate the possible origins of this specialization, T4moC, mutated variants of T4moC, and three other purified ferredoxins (the Thermus Rieske protein, the Burkholderia cepacia Rieske-type biphenyl dioxygenase ferredoxin BphF, and the Ralstonia pickettii PK01 toluene monooxygenase TbuB, the Rieske-type ferredoxin from another diiron monooxygenase complex) were studied by redox potential measurements and their ability to complement the catalytic function of the reconstituted toluene 4-monooxygenase complex. A saturation mutagenesis of T4moC W69 indicates that an aromatic residue may modulate the redox potential and is also necessary for activity and/or stability. The redox potential of T4moC was determined to be -173 mV, W69F T4moC was -139 mV, and TbuB was -150 mV. For comparison, BphF had a redox potential of -157 mV [Couture et al. (2001) Biochemistry 40, 84-92]. Of these ferredoxins, all except BphF were able to provide catalytic activity. Given the range in redox potentials observed in the active ferredoxins, shape and electrostatics are strongly implicated in the catalytic specialization. Mutagenesis of other T4moC surface residues gave further insight into possible origins of catalytic specialization. Thus R65A T4moC gave an alteration in apparent KM only, while D82A/D83A T4moC gave alterations in both apparent kcat and KM. Since the different catalytic results were obtained by mutagenesis of residues lying on different sides of the protein adjacent to the [2Fe-2S] cluster, the results suggest that two different faces of T4moC may be involved in protein-protein interactions during catalysis.

In nature, bacteria and fungi are able to utilize recalcitrant plant materials by secreting a diverse set of enzymes. While genomic sequencing efforts offer exhaustive lists of genes annotated as potential polysaccharide-degrading enzymes, biochemical and functional characterizations of the encoded proteins are still needed to realize the full potential of this natural genomic diversity. This chapter outlines an application of wheat germ cell-free translation to the study of biofuel enzymes using genes from Clostridium thermocellum, a model cellulolytic organism. Since wheat germ extract lacks enzymatic activities that can hydrolyze insoluble polysaccharide substrates and is likewise devoid of enzymes that consume the soluble sugar products, the cell-free translation reactions provide a clean background for production and study of the reactions of biofuel enzymes. Examples of assays performed with individual enzymes or with small sets of enzymes obtained directly from cell-free translation are provided.

The cooperative assembly of FtsZ, the prokaryotic homologue of tubulin, plays an essential role in cell division. FtsZ is a potential drug target, as illustrated by the small-molecule cell-cycle inhibitor and antibacterial agent PC190723 that targets FtsZ. We demonstrate that PC190723 negatively modulates Staphylococcus aureus FtsZ polymerization cooperativity as reflected in polymerization at lower concentrations without a defined critical concentration. The crystal structure of the S. aureus FtsZ-PC190723 complex shows a domain movement that would stabilize the FtsZ protofilament over the monomeric state, with the conformational change mediated from the GTP-binding site to the C-terminal domain via helix 7. Together, the results reveal the molecular mechanism of FtsZ modulation by PC190723 and a conformational switch to the high-affinity state that enables polymer assembly.

A microarray study of chemostat growth on insoluble cellulose or soluble cellobiose has provided substantial new information on Clostridium thermocellum gene expression. This is the first comprehensive examination of gene expression in C. thermocellum under defined growth conditions. Expression was detected from 2,846 of 3,189 genes, and regression analysis revealed 348 genes whose changes in expression patterns were growth rate and/or substrate dependent. Successfully modeled genes included those for scaffoldin and cellulosomal enzymes, intracellular metabolic enzymes, transcriptional regulators, sigma factors, signal transducers, transporters, and hypothetical proteins. Unique genes encoding glycolytic pathway and ethanol fermentation enzymes expressed at high levels simultaneously with previously established maximal ethanol production were also identified. Ranking of normalized expression intensities revealed significant changes in transcriptional levels of these genes. The pattern of expression of transcriptional regulators, sigma factors, and signal transducers indicates that response to growth rate is the dominant global mechanism used for control of gene expression in C. thermocellum.

Despite the need for new antibiotics to treat drug-resistant bacteria, current clinical combinations are largely restricted to β-lactam antibiotics paired with β-lactamase inhibitors. We have adapted a Staphylococcus aureus antisense knockdown strategy to genetically identify the cell division Z ring components-FtsA, FtsZ, and FtsW-as β-lactam susceptibility determinants of methicillin-resistant S. aureus (MRSA). We demonstrate that the FtsZ-specific inhibitor PC190723 acts synergistically with β-lactam antibiotics in vitro and in vivo and that this combination is efficacious in a murine model of MRSA infection. Fluorescence microscopy localization studies reveal that synergy between these agents is likely to be elicited by the concomitant delocalization of their cognate drug targets (FtsZ and PBP2) in MRSA treated with PC190723. A 2.0 A crystal structure of S. aureus FtsZ in complex with PC190723 identifies the compound binding site, which corresponds to the predominant location of mutations conferring resistance to PC190723 (PC190723(R)). Although structural studies suggested that these drug resistance mutations may be difficult to combat through chemical modification of PC190723, combining PC190723 with the β-lactam antibiotic imipenem markedly reduced the spontaneous frequency of PC190723(R) mutants. Multiple MRSA PC190723(R) FtsZ mutants also displayed attenuated virulence and restored susceptibility to β-lactam antibiotics in vitro and in a mouse model of imipenem efficacy. Collectively, these data support a target-based approach to rationally develop synergistic combination agents that mitigate drug resistance and effectively treat MRSA infections.