Drug screening approach against mycobacterial fatty acyl-AMP ligase FAAL32 renews the interest of the salicylanilide pharmacophore in the fight against tuberculosis.

Tuberculosis (TB) remains a global health crisis, further exacerbated by the slow pace of new treatment options, and the emergence of extreme and total drug resistance to existing drugs. The challenge to developing new antibacterial compounds with activity against Mycobacterium tuberculosis (Mtb), the causative agent of TB, is in part due to unique features of this pathogen, especially the composition and structure of its complex cell envelope. Therefore, targeting enzymes involved in cell envelope synthesis has been of major interest for anti-TB drug discovery. FAAL32 is a fatty acyl-AMP ligase involved in the biosynthesis of the cell wall mycolic acids, and a potential target for drug discovery. To rapidly advance research in this area, we initiated a drug repurposing campaign and screened a collection of 1280 approved human or veterinary drugs (Prestwick Chemical Library) using a biochemical assay that reads out FAAL32 inhibition. These efforts led to the discovery of salicylanilide closantel, and some of its derivatives as inhibitors with potent in vitro activity against M. tuberculosis. These results suggest that salicylanilide represents a potentially promising pharmacophore for the conception of novel anti-tubercular candidates targeting FAAL32 that would open new targeting opportunities. Moreover, this work illustrates the value of drug repurposing campaigns to discover new leads in challenging drug discovery fields.

Liposome-Based Methods to Study Protein-Phosphoinositide Interaction.

Following their generation by lipid kinases and phosphatases, phosphoinositides regulate important biological processes such as cytoskeleton rearrangement, membrane remodeling/trafficking, and gene expression through the interaction of their phosphorylated inositol head group with a variety of protein domains such as PH, PX, and FYVE. Therefore, it is important to determine the specificity of phosphoinositides toward effector proteins to understand their impact on cellular physiology. Several methods have been developed to identify and characterize phosphoinositide effectors, and liposomes-based methods are preferred because the phosphoinositides are incorporated in a membrane, the composition of which can mimic cellular membranes. In this report, we describe the experimental setup for liposome flotation assay and a recently developed method called protein-lipid interaction by fluorescence (PLIF) for the characterization of phosphoinositide-binding specificities of proteins.

Molecular Basis for Extender Unit Specificity of Mycobacterial Polyketide Synthases.

Mycobacterium tuberculosis is the causative agent of the tuberculosis disease, which claims more human lives each year than any other bacterial pathogen. M. tuberculosis and other mycobacterial pathogens have developed a range of unique features that enhance their virulence and promote their survival in the human host. Among these features lies the particular cell envelope with high lipid content, which plays a substantial role in mycobacterial pathogenicity. Several envelope components of M. tuberculosis and other mycobacteria, e.g., mycolic acids, phthiocerol dimycocerosates, and phenolic glycolipids, belong to the "family" of polyketides, secondary metabolites synthesized by fascinating versatile enzymes-polyketide synthases. These megasynthases consist of multiple catalytic domains, among which the acyltransferase domain plays a key role in selecting and transferring the substrates required for polyketide extension. Here, we present three new crystal structures of acyltransferase domains of mycobacterial polyketide synthases and, for one of them, provide evidence for the identification of residues determining extender unit specificity. Unravelling the molecular basis for such specificity is of high importance considering the role played by extender units for the final structure of key mycobacterial components. This work provides major advances for the use of mycobacterial polyketide synthases as potential therapeutic targets and, more generally, contributes to the prediction and bioengineering of polyketide synthases with desired specificity.

PLIF: A rapid, accurate method to detect and quantitatively assess protein-lipid interactions.

Phosphoinositides are a type of cellular phospholipid that regulate signaling in a wide range of cellular and physiological processes through the interaction between their phosphorylated inositol head group and specific domains in various cytosolic proteins. These lipids also influence the activity of transmembrane proteins. Aberrant phosphoinositide signaling is associated with numerous diseases, including cancer, obesity, and diabetes. Thus, identifying phosphoinositide-binding partners and the aspects that define their specificity can direct drug development. However, current methods are costly, time-consuming, or technically challenging and inaccessible to many laboratories. We developed a method called PLIF (for "protein-lipid interaction by fluorescence") that uses fluorescently labeled liposomes and tethered, tagged proteins or peptides to enable fast and reliable determination of protein domain specificity for given phosphoinositides in a membrane environment. We validated PLIF against previously known phosphoinositide-binding partners for various proteins and obtained relative affinity profiles. Moreover, PLIF analysis of the sorting nexin (SNX) family revealed not only that SNXs bound most strongly to phosphatidylinositol 3-phosphate (PtdIns3P or PI3P), which is known from analysis with other methods, but also that they interacted with other phosphoinositides, which had not previously been detected using other techniques. Different phosphoinositide partners, even those with relatively weak binding affinity, could account for the diverse functions of SNXs in vesicular trafficking and protein sorting. Because PLIF is sensitive, semiquantitative, and performed in a high-throughput manner, it may be used to screen for highly specific protein-lipid interaction inhibitors.

Probing the Functions of Carbohydrate Binding Modules in the CBEL Protein from the Oomycete Phytophthora parasitica.

Oomycetes are microorganisms that are distantly related to true fungi and many members of this phylum are major plant pathogens. Oomycetes express proteins that are able to interact with plant cell wall polysaccharides, such as cellulose. This interaction is thought to be mediated by carbohydrate-binding modules that are classified into CBM family 1 in the CAZy database. In this study, the two CBMs (1-1 and 1-2) that form part of the cell wall glycoprotein, CBEL, from Phytophthora parasitica have been submitted to detailed characterization, first to better quantify their interaction with cellulose and second to determine whether these CBMs can be useful for biotechnological applications, such as biomass hydrolysis. A variety of biophysical techniques were used to study the interaction of the CBMs with various substrates and the data obtained indicate that CBEL's CBM1-1 exhibits much greater cellulose binding ability than CBM1-2. Engineering of the family 11 xylanase from Talaromyces versatilis (TvXynB), an enzyme that naturally bears a fungal family 1 CBM, has produced two variants. The first one lacks its native CBM, whereas the second contains the CBEL CBM1-1. The study of these enzymes has revealed that wild type TvXynB binds to cellulose, via its CBM1, and that the substitution of its CBM by oomycetal CBM1-1 does not affect its activity on wheat straw. However, intriguingly the addition of CBEL during the hydrolysis of wheat straw actually potentiates the action of TvXynB variant lacking a CBM1. This suggests that the potentiating effect of CBM1-1 might not require the formation of a covalent linkage to TvXynB.

New insights on the mechanism of quinoline-based DNA Methyltransferase inhibitors.

Among the epigenetic marks, DNA methylation is one of the most studied. It is highly deregulated in numerous diseases, including cancer. Indeed, it has been shown that hypermethylation of tumor suppressor genes promoters is a common feature of cancer cells. Because DNA methylation is reversible, the DNA methyltransferases (DNMTs), responsible for this epigenetic mark, are considered promising therapeutic targets. Several molecules have been identified as DNMT inhibitors and, among the non-nucleoside inhibitors, 4-aminoquinoline-based inhibitors, such as SGI-1027 and its analogs, showed potent inhibitory activity. Here we characterized the in vitro mechanism of action of SGI-1027 and two analogs. Enzymatic competition studies with the DNA substrate and the methyl donor cofactor, S-adenosyl-l-methionine (AdoMet), displayed AdoMet non-competitive and DNA competitive behavior. In addition, deviations from the Michaelis-Menten model in DNA competition experiments suggested an interaction with DNA. Thus their ability to interact with DNA was established; although SGI-1027 was a weak DNA ligand, analog 5, the most potent inhibitor, strongly interacted with DNA. Finally, as 5 interacted with DNMT only when the DNA duplex was present, we hypothesize that this class of chemical compounds inhibit DNMTs by interacting with the DNA substrate.

The high resolution structure of tyrocidine A reveals an amphipathic dimer.

Tyrocidine A, one of the first antibiotics ever to be discovered, is a cyclic decapeptide that binds to membranes of target bacteria, disrupting their integrity. It is active against a broad spectrum of Gram-positive organisms, and has recently engendered interest as a potential scaffold for the development of new drugs to combat antibiotic-resistant pathogens. We present here the X-ray crystal structure of tyrocidine A at a resolution of 0.95Å. The structure reveals that tyrocidine forms an intimate and highly amphipathic homodimer made up of four beta strands that associate into a single, highly curved antiparallel beta sheet. We used surface plasmon resonance and potassium efflux assays to demonstrate that tyrocidine binds tightly to mimetics of bacterial membranes with an apparent dissociation constant (K(D)) of 10 µM, and efficiently permeabilizes bacterial cells at concentrations equal to and below the K(D). Using variant forms of tyrocidine in which the fluorescent probe p-cyano-phenylalanine had been inserted on either the polar or apolar face of the molecule, we performed fluorescence quenching experiments, using both water-soluble and membrane-embedded quenchers. The quenching results, together with the structure, strongly support a membrane association model in which the convex, apolar face of tyrocidine's beta sheet is oriented toward the membrane interior, while the concave, polar face is presented to the aqueous phase.

First structural insights into α-L-arabinofuranosidases from the two GH62 glycoside hydrolase subfamilies.

α-L-arabinofuranosidases are glycoside hydrolases that specifically hydrolyze non-reducing residues from arabinose-containing polysaccharides. In the case of arabinoxylans, which are the main components of hemicellulose, they are part of microbial xylanolytic systems and are necessary for complete breakdown of arabinoxylans. Glycoside hydrolase family 62 (GH62) is currently a small family of α-L-arabinofuranosidases that contains only bacterial and fungal members. Little is known about the GH62 mechanism of action, because only a few members have been biochemically characterized and no three-dimensional structure is available. Here, we present the first crystal structures of two fungal GH62 α-L-arabinofuranosidases from the basidiomycete Ustilago maydis (UmAbf62A) and ascomycete Podospora anserina (PaAbf62A). Both enzymes are able to efficiently remove the α-L-arabinosyl substituents from arabinoxylan. The overall three-dimensional structure of UmAbf62A and PaAbf62A reveals a five-bladed ß-propeller fold that confirms their predicted classification into clan GH-F together with GH43 α-L-arabinofuranosidases. Crystallographic structures of the complexes with arabinose and cellotriose reveal the important role of subsites +1 and +2 for sugar binding. Intriguingly, we observed that PaAbf62A was inhibited by cello-oligosaccharides and displayed binding affinity to cellulose although no activity was observed on a range of cellulosic substrates. Bioinformatic analyses showed that UmAbf62A and PaAbf62A belong to two distinct subfamilies within the GH62 family. The results presented here provide a framework to better investigate the structure-function relationships within the GH62 family.

A carrier protein strategy yields the structure of dalbavancin.

Many large natural product antibiotics act by specifically binding and sequestering target molecules found on bacterial cells. We have developed a new strategy to expedite the structural analysis of such antibiotic-target complexes, in which we covalently link the target molecules to carrier proteins, and then crystallize the entire carrier-target-antibiotic complex. Using native chemical ligation, we have linked the Lys-D-Ala-D-Ala binding epitope for glycopeptide antibiotics to three different carrier proteins. We show that recognition of this peptide by multiple antibiotics is not compromised by the presence of the carrier protein partner, and use this approach to determine the first-ever crystal structure for the new therapeutic dalbavancin. We also report the first crystal structure of an asymmetric ristocetin antibiotic dimer, as well as the structure of vancomycin bound to a carrier-target fusion. The dalbavancin structure reveals an antibiotic molecule that has closed around its binding partner; it also suggests mechanisms by which the drug can enhance its half-life by binding to serum proteins, and be targeted to bacterial membranes. Notably, the carrier protein approach is not limited to peptide ligands such as Lys-D-Ala-D-Ala, but is applicable to a diverse range of targets. This strategy is likely to yield structural insights that accelerate new therapeutic development.

Structure of ristocetin A in complex with a bacterial cell-wall mimetic.

Antimicrobial drug resistance is a serious public health problem and the development of new antibiotics has become an important priority. Ristocetin A is a class III glycopeptide antibiotic that is used in the diagnosis of von Willebrand disease and which has served as a lead compound for the development of new antimicrobial therapeutics. The 1.0 A resolution crystal structure of the complex between ristocetin A and a bacterial cell-wall peptide has been determined. As is observed for most other glycopeptide antibiotics, it is shown that ristocetin A forms a back-to-back dimer containing concave binding pockets that recognize the cell-wall peptide. A comparison of the structure of ristocetin A with those of class I glycopeptide antibiotics such as vancomycin and balhimycin identifies differences in the details of dimerization and ligand binding. The structure of the ligand-binding site reveals a likely explanation for ristocetin A's unique anticooperativity between dimerization and ligand binding.

Nuclear receptor ligand-binding domains: reduction of helix H12 dynamics to favour crystallization.

Crystallization trials of the human retinoid X receptor alpha ligand-binding domain (RXRalpha LBD) in complex with various ligands have been carried out. Using fluorescence anisotropy, it has been found that when compared with agonists these small-molecule effectors enhance the dynamics of the RXRalpha LBD C-terminal helix H12. In some cases, the mobility of this helix could be dramatically reduced by the addition of a 13-residue co-activator fragment (CoA). In keeping with these observations, crystals have been obtained of the corresponding ternary RXRalpha LBD-ligand-CoA complexes. In contrast, attempts to crystallize complexes with a highly mobile H12 remained unsuccessful. These experimental observations substantiate the previously recognized role of co-regulator fragments in facilitating the crystallization of nuclear receptor LBDs.

N-1H-benzimidazol-5-ylbenzenesulfonamide derivatives as potent hPXR agonists.

The Human Pregnane X Receptor (hPXR) is a nuclear receptor that regulates the expression of phase I and phase II drug-metabolizing enzymes, as well as that of drug transporters. Because this receptor plays a critical role in protecting tissues from potentially toxic endo- and xenobiotics, highly active agonists could represent novel therapeutic tools in treating several human diseases. Using an in vitro screening reporter system that allow to characterize hPXR activators and a first step of chemical modifications of an original agonist ligand (C2BA-4, 1-(2-chlorophenyl)-N-[1-(1-phenylethyl)-1H-benzimidazol-5-yl]methanesulfonamide), we identified compounds with a N-1H-benzimidazol-5-ylbenzenesulfonamide scaffold as a potent family of hPXR agonists. Further chemical modifications allowed us to identify enhanced activators, notably N-(1-benzyl-1H-benzimidazol-5-yl)-2,3,4,5,6-pentamethylbenzenesulfonamide (6n) with an EC(50) value in the subnanomolar range. Accordingly to their potent EC(50), these compounds induced an efficient protection of hPXR against proteolytic digestion by trypsin even at very low ligand concentrations and were able to induce the expression of the main target genes of hPXR, CYP3A4 and CYP2B6, in primary cultures of human hepatocytes.

Modulators of the structural dynamics of the retinoid X receptor to reveal receptor function.

Retinoid X receptors (RXRalpha, -beta, and -gamma) occupy a central position in the nuclear receptor superfamily, because they form heterodimers with many other family members and hence are involved in the control of a variety of (patho)physiologic processes. Selective RXR ligands, referred to as rexinoids, are already used or are being developed for cancer therapy and have promise for the treatment of metabolic diseases. However, important side effects remain associated with existing rexinoids. Here we describe the rational design and functional characterization of a spectrum of RXR modulators ranging from partial to pure antagonists and demonstrate their utility as tools to probe the implication of RXRs in cell biological phenomena. One of these ligands renders RXR activity particularly sensitive to coactivator levels and has the potential to act as a cell-specific RXR modulator. A combination of crystallographic and fluorescence anisotropy studies reveals the molecular details accounting for the agonist-to-antagonist transition and provides direct experimental evidence for a correlation between the pharmacological activity of a ligand and its impact on the structural dynamics of the activation helix H12. Using RXR and its cognate ligands as a model system, our correlative analysis of 3D structures and dynamic data provides an original view on ligand actions and enables the establishment of mechanistic concepts, which will aid in the development of selective nuclear receptor modulators.

Discovery of a highly active ligand of human pregnane x receptor: a case study from pharmacophore modeling and virtual screening to "in vivo" biological activity.

The human pregnane X receptor (hPXR) is a nuclear receptor that regulates the expression of phase I and II drug-metabolizing enzymes as well as that of drug transporters. In addition, this receptor plays a critical role in cholesterol homeostasis and in protecting tissues from potentially toxic endobiotics. hPXR is activated by a broad spectrum of low-affinity compounds including xenobiotics and endobiotics such as bile acids and their precursors. Crystallographic studies revealed a ligand binding domain (LBD) with a large and conformable binding pocket that is likely to contribute to the ability of hPXR to respond to compounds of varying size and shape. Here, we describe an in silico method that allowed the identification of nine novel hPXR agonists. We further characterize the compound 1-(2-chlorophenyl)-N-[1-(1-phenylethyl)-1H-benzimidazol-5-yl]methanesulfonamide (C2BA-4), a methanesulfonamide that activates PXR specifically and more potently than does the reference compound 4-[2,2-bis(diethoxyphosphoryl)ethenyl]-2,6-ditert-butyl-phenol (SR12813) in our stable cell line expressing a Gal4-PXR and a GAL4 driven luciferase reporter gene. Furthermore treatment of primary human hepatocytes with C2BA-4 results in a marked induction of the mRNA expression of hPXR target genes, such as cytochromes P450 3A4 and 2B6. Finally, C2BA-4 is also able to induce hPXR-mediated in vivo luciferase expression in HGPXR stable bioluminescent cells implanted in mice. The study suggests new directions for the rational design of selective hPXR agonists and antagonists.

Androgen and estrogen receptors: potential of crystallography in the fight against cancer.

Androgen (AR) and estrogen (ERalpha and ERbeta) receptors are primary targets in the treatment of hormone-sensitive tumors such as prostate or breast cancers. Because of their diverse and important roles in normal and pathologic physiology, these nuclear receptors have prompted intense research. Here, we review how structural studies conducted over the past several years on AR and ERs have provided significant advances in our comprehension of androgen and estrogen signaling and how this information can be used in the fight against cancer.

Molecular basis of the effects of chloride ion on the acid-base catalyst in the mechanism of pancreatic alpha-amylase.

Pig pancreatic alpha-amylase (PPA), an enzyme belonging to the alpha-amylase family, is involved in the degradation of starch. Like some other members of this family, PPA requires chloride to reach maximum activity levels. To further explain the mechanism of chloride activation, a crystal of wild-type PPA soaked with maltopentaose using a chloride-free buffer was analyzed by X-ray crystallography. A conspicuous reorientation of the acid/base catalyst Glu233 residue was found to occur. The structural results, along with kinetic data, show that the acid/base catalyst is maintained in the active site, in an optimum position, pointing toward the scissile bond-atom, due to the presence of chloride ions. The present study therefore explains the mechanism of PPA activation by chloride ions.

How estrogen-specific proteins discriminate estrogens from androgens: a common steroid binding site architecture.

Steroid hormones play an essential role in a wide range of physiological and pathological processes, such as growth, metabolism, aging, and hormone-sensitive cancers. Estrogens are no exception and influence growth, differentiation, and functioning of many target tissues, such as the mammary gland, uterus, hypothalamus, pituitary, bone, and liver. Although very similar in structure, each steroid class (i.e., estrogens, androgens, progestins, mineral corticoids, or glucocorticoids) is responsible for distinct physiological processes. To permit specific biological responses for a given steroid class, specific proteins are responsible for steroid bioactivation, action, and inactivation, yet they have low or no affinity to other classes. Estrogens make no exception and possess their own set of related proteins. To understand the molecular basis underlying estrogen recognition from other steroids, structural features of estrogen-specific proteins were analyzed along with their ability to discriminate between steroid hormones belonging to different classes. Hence, the study of all estrogen-specific proteins for which an atomic structure has been determined demonstrated that a common steroid-binding pocket architecture is shared by these proteins. This architecture is composed of the following elements: i) a glutamate residue acting as a proton acceptor coupled with a proton donor that interact with the steroid O3; ii) a proton donor (His or Ser) that interacts with O17; iii) a highly conserved sandwich-like structure providing steric hindrance and preventing C19 steroid from binding; and iv) several amino acid residues interacting with the C18. As these different estrogen-specific proteins are not related in overall sequence, the inference is that the steroid binding site in these proteins has originated by convergent evolution.

Pseudo-symmetry of C19 steroids, alternative binding orientations, and multispecificity in human estrogenic 17beta-hydroxysteroid dehydrogenase.

Steroids are implicated in many physiological processes, such as reproduction, aging, metabolism, and cancer. To understand the molecular basis for steroid recognition and discrimination, we studied the human estrogenic 17beta-hydroxysteroid dehydrogenase (17beta-HSD1) responsible for the last step in the bioactivation of all estrogens. Here we report the first observation of the conversion of dihydrotestosterone (DHT) into 3beta,17beta-androstanediol (3beta-diol) by 17beta-HSD1, an estrogenic enzyme studied for more than half a century. Kinetic observations demonstrate that both the 3beta-reduction of DHT into 3beta-diol (kcat = 0.040 s(-1)1; Km = 32 +/- 9 microM) and the 17beta-oxidation of DHT into androstandione (A-dione) (kcat = 0.19 s(-1); Km = 26 +/-6 microM) are catalyzed by 17beta-HSD1 via alternative binding orientation of the steroid. The reduction of DHT was also observed in intact cells by using HEK-293 cells stably transformed with 17beta-HSD1. The high-resolution structure of a 17beta-HSD1-C19-steroid (testosterone) complex solved at 1.54 A demonstrates that the steroid is reversibly oriented in the active site, which strongly supports the existence of alternative binding mode. Such a phenomenon can be explained by the pseudo-symmetric structure of C19-steroids. Our results confirm the role of the Leu149 residue in C18/C19-steroid discrimination and suggest a possible mechanism of 17beta-HSD1 in the modulation of DHT levels in tissues, such as the breast, where both the enzyme and DHT are present.