Label-free affinity screening, design and synthesis of inhibitors targeting the Mycobacterium tuberculosis L-alanine dehydrogenase.

The ability of Mycobacterium tuberculosis (Mtb) to persist in its host may enable an evolutionary advantage for drug resistant variants to emerge. A potential strategy to prevent persistence and gain drug efficacy is to directly target the activity of enzymes that are crucial for persistence. We present a method for expedited discovery and structure-based design of lead compounds by targeting the hypoxia-associated enzyme L-alanine dehydrogenase (AlaDH). Biochemical and structural analyses of AlaDH confirmed binding of nucleoside derivatives and showed a site adjacent to the nucleoside binding pocket that can confer specificity to putative inhibitors. Using a combination of dye-ligand affinity chromatography, enzyme kinetics and protein crystallographic studies, we show the development and validation of drug prototypes. Crystal structures of AlaDH-inhibitor complexes with variations at the N6 position of the adenyl-moiety of the inhibitor provide insight into the molecular basis for the specificity of these compounds. We describe a drug-designing pipeline that aims to block Mtb to proliferate upon re-oxygenation by specifically blocking NAD accessibility to AlaDH. The collective approach to drug discovery was further evaluated through in silico analyses providing additional insight into an efficient drug development strategy that can be further assessed with the incorporation of in vivo studies.

Anti-Microbial Activity of Aliphatic Alcohols from Chinese Black Cardamom (Amomum tsao-ko) against Mycobacterium tuberculosis H37Rv

The fruits of Amomun tsao-ko (Chinese black cardamom; Zingiberaceae) contain an abundance of essential oils, which have previously demonstrated significant antimicrobial activity. In our preliminary search for natural anti-tuberculosis agents, an acetone extract of A. tsao-ko (AAE) exhibited strong antibacterial activity against Mycobacterium tuberculosis H37Rv. Therefore, the aim of this study was to find the principal compounds in an AAE against M. tuberculosis. Nine aliphatic compounds (1−9) including a new compound (1, tsaokol B) and a new natural unsaturated aliphatic diester (6), together with three acyclic terpenoids (10−12), were isolated from an AAE by repetitive chromatography. The structures of the isolates were determined by spectroscopic data analysis. All isolates were evaluated for activity against M. tuberculosis H37Rv. Isolated compounds 1−6, and 11 had MICs ranging from 0.6−89 µg/mL. In contrast, compounds 7 to 10, and 12 had MICs that were >100 µg/mL. Tsaokol A (3) was the most active compound with MICs of 0.6 µg/mL and 1.4 µg/mL, respectively, against replicating and nonreplicating M. tuberculosis. These results are the first to illustrate the potency of tsaokol A (3) as a natural drug candidate with good selectivity for treating tuberculosis.

In Vitro Profiling of Antitubercular Compounds by Rapid, Efficient, and Nondestructive Assays Using Autoluminescent Mycobacterium tuberculosis.

Anti-infective drug discovery is greatly facilitated by the availability of in vitro assays that are more proficient at predicting the preclinical success of screening hits. Tuberculosis (TB) drug discovery is hindered by the relatively slow growth rate of Mycobacterium tuberculosis and the use of whole-cell-based in vitro assays that are inherently time-consuming, and for these reasons, rapid, noninvasive bioluminescence-based assays have been widely used in anti-TB drug discovery and development. In this study, in vitro assays that employ autoluminescent M. tuberculosis were optimized to determine MIC, minimum bactericidal concentration (MBC), time-kill curves, activity against macrophage internalized M. tuberculosis (90% effective concentration [EC90]), and postantibiotic effect (PAE) to provide rapid and dynamic biological information. Standardization of the luminescence-based MIC, MBC, time-kill, EC90, and PAE assays was accomplished by comparing results of established TB drugs and two ClpC1-targeting TB leads, ecumicin and rufomycin, to those obtained from conventional assays and/or to previous studies. Cumulatively, the use of the various streamlined luminescence-based in vitro assays has reduced the time for comprehensive in vitro profiling (MIC, MBC, time-kill, EC90, and PAE) by 2 months. The luminescence-based in vitro MBC and EC90 assays yield time and concentration-dependent kill information that can be used for pharmacokinetic-pharmacodynamic (PK-PD) modeling. The MBC and EC90 time-kill graphs revealed a significantly more rapid bactericidal activity for ecumicin than rufomycin. The PAEs of both ecumicin and rufomycin were comparable to that of the first-line TB drug rifampin. The optimization of several nondestructive, luminescence-based TB assays facilitates the in vitro profiling of TB drug leads in an efficient manner.

New tuberculosis drug targets, their inhibitors, and potential therapeutic impact.

The current tuberculosis (TB) predicament poses numerous challenges and therefore every incremental scientific work and all positive socio-political engagements, are steps taken in the right direction to eradicate TB. Progression of the late stage TB-drug pipeline into the clinics is an immediate deliverable of this global effort. At the same time, fueling basic research and pursuing early discovery work must be sustained to maintain a healthy TB-drug pipeline. This review encompasses a broad analysis of chemotherapeutic strategies that target the DNA replication, protein synthesis, cell wall biosynthesis, energy metabolism and proteolysis of Mycobacterium tuberculosis (Mtb). It includes a status check of the current TB-drug pipeline with a focus on the associated biology, emerging targets, and their promising chemical inhibitors. Potential synergies and/or gaps within or across different chemotherapeutic strategies are systematically reviewed as well.

Synthetic analogs of rhamnolipids modulate structured biofilms formed by rhamnolipid-nonproducing mutant of Pseudomonas aeruginosa.

Rhamnolipids secreted by Pseudomonas aeruginosa are required for the bacteria to form biofilm efficiently and form biofilm with internal structures including pores and channels. In this work, we explore the effect of a class of synthetic analogs of rhamnolipids at controlling (promoting and inhibiting) the biofilm formation activities of a non-rhamnolipid-producing strain - rhlA - of P. aeruginosa. This class of rhamnolipid analogs is known to modulate the swarming motilities of wild-type PAO1 and rhlA mutant, but its effect on biofilm formation of rhlA mutant is unknown. We show that small structural details of these molecules are important for the bioactivities, but do not affect the general physical properties of the molecules. The bioactive synthetic analogs of rhamnolipids promote biofilm formation by rhlA mutant at low concentrations, but inhibit the biofilm formation at high concentrations. To explore the internal structures formed by the biofilms, we first demonstrate that wild-type biofilms are formed with substantial topography (hills and valleys) when the sample is under shaking conditions. Using this observation as a comparison, we found that synthetic analogs of rhamnolipids promoted structured (porous) biofilm of rhlA mutant, at intermediate concentrations between the low ones that promoted biofilm formation and the high ones that inhibited biofilm formation. This study suggests a potential chemical signaling approach to control multiple bacterial activities.

Chemical Signals of Synthetic Disaccharide Derivatives Dominate Rhamnolipids at Controlling Multiple Bacterial Activities.

Microbes secrete molecules that modify their environment. Here, we demonstrate a class of synthetic disaccharide derivatives (DSDs) that mimics and dominates the activity of naturally secreted rhamnolipids by Pseudomonas aeruginosa. The DSDs exhibit the dual function of activating and inhibiting the swarming motility through a concentration-dependent activity reversal that is characteristic of signaling molecules. Whereas DSDs tethered with a saturated farnesyl group exhibit inhibition of both biofilm formation and swarming motility, with higher activities than rhamnolipids, a saturated farnesyl tethered with a sulfonate group only inhibits swarming motility but promote biofilm formation. These results identified important structural elements for controlling swarming motility, biofilm formation, and bacterial adhesion and suggest an effective chemical approach to control intertwined signaling processes that are important for biofilm formation and motilities.

Specific maltose derivatives modulate the swarming motility of nonswarming mutant and inhibit bacterial adhesion and biofilm formation by Pseudomonas aeruginosa.

We have demonstrated that specific synthetic maltose derivatives activate the swarming motility of a Pseudomonas aeruginosa nonswarming mutant (rhlA) at low concentration, but inhibit it at high concentration. Although these molecules are not microbicidal, active maltose derivatives with bulky hydrocarbon groups inhibited bacterial adhesion, and exhibited biofilm inhibition and dispersion (IC50 ~20 µM and DC50 ~30 µM, respectively). Because the swarming motility of the rhlA mutant is abolished by the lack natural rhamnolipids, the swarming activation suggests that maltose derivatives are analogues of rhamnolipids. Together, these results suggest a new approach of controlling multiple bacterial activities (bacterial adhesion, biofilm formation, and swarming motility) by a set of disaccharide-based molecules.

Bicyclic brominated furanones: a new class of quorum sensing modulators that inhibit bacterial biofilm formation.

Both natural and synthetic brominated furanones are known to inhibit biofilm formation by bacteria, but their toxicity to mammalian cells is often not reported. Here, we designed and synthesized a new class of brominated furanones (BBFs) that contained a bicyclic structure having one bromide group with well-defined regiochemistry. This class of molecules exhibited reduction in the toxicity to mammalian cells (human neuroblastoma SK-N-SH) and did not inhibit bacteria (Pseudomonas aeruginosa and Escherichia coli) growth, but retained the inhibitory activity towards biofilm formation of bacteria. In addition, all the BBFs inhibited the production of virulence factor elastase B in P. aeruginosa. To explore the effect of BBFs on quorum sensing, we used a reporter gene assay and found that 6-BBF and 7-BBF exhibited antagonistic activities for LasR protein in the lasI quorum sensing circuit, while 5-BBF showed agonistic activity for the rhlI quorum sensing circuit. This study suggests that structural variation of brominated furanones can be designed for targeted functions to control biofilm formation.

The ability of single-chain surfactants to emulsify an aqueous-based liquid crystal oscillates with odd-even parity of alkyl-chain length.

The physical properties of many organic molecules often oscillate when the number of carbons in their aliphatic chains changes from odd to even. This odd-even effect for single-chain surfactants in solution is rarely observed. Here, we report the ability of single-chain surfactants to emulsify a class of non-amphiphilic organic salts, disodium cromoglycate (5'DSCG) oscillates as a function of the odd or even number of the aliphatic carbons. This system provides a water-in-oil-in-water emulsion, in which aqueous droplets of 5'DSCG in liquid crystal phases are coated with single-chain surfactants in a bulk carrying aqueous solution. For both surfactants of [Formula: see text] and CH3(CH2)nCOO(-)Na(+), the ability to emulsify 5'DSCG molecules in water is stronger for surfactants with an odd number of sp(3)-hybridized carbon atoms in the aliphatic chains than those with an even number. This observed odd-even effect is consistent with the notion that conventional micelles possess a core of randomly arranged surfactant hydrocarbon tails. However, this water-in-oil-in-water resembles a vesicle system in which the surfactants assemble in a highly ordered structure that separates two aqueous systems. These new self-assembled phases have potential application in the formulation and design of new organic soft materials.

Emulsion of aqueous-based nonspherical droplets in aqueous solutions by single-chain surfactants: templated assembly by nonamphiphilic lyotropic liquid crystals in water.

Single-chain surfactants usually emulsify and stabilize oily substances into droplets in an aqueous solution. Here, we report a coassembly system, in which single types of anionic or non-ionic surfactants emulsify a class of water-soluble nonamphiphilic organic salts with fused aromatic rings in aqueous solutions. The nonamphiphilic organic salts are in turn promoted to form droplets of water-based liquid crystals (chromonic liquid crystals) encapsulated by single-chain surfactants. The droplets, stabilized against coalescence by encapsulated in a layer (or layers) of single chain surfactants, are of both nonspherical tactoid (elongated ellipsoid with pointy ends) and spherical shapes. The tactoids have an average long axis of ∼9 µm and a short axis of ∼3.5 µm with the liquid crystal aligning parallel to the droplet surface. The spherical droplets are 5-10 µm in diameter and have the liquid crystal aligning perpendicular to the droplet surface and a point defect in the center. Cationic and zwitterionic surfactants studied in this work did not promote the organic salt to form droplets. These results illustrate the complex interplay of self-association and thermodynamic incompatibility of molecules in water, which can cause new assembly behavior, including potential formation of vesicles or other assemblies, from surfactants that usually form only micelles. These unprecedented tactoidal shaped droplets also provide potential for the fabrication of new soft organic microcapsules.

Noncovalent polymerization of mesogens crystallizes lysozyme: correlation between nonamphiphilic lyotropic liquid crystal phase and protein crystal formation.

Crystallization of proteins is important for fundamental studies and biopharmaceutical development but remains largely an empirical science. Here, we report the use of organic salts that can form a class of unusual nonamphiphilic lyotropic liquid crystals to crystallize the protein lysozyme. Certain nonamphiphilic organic molecules with fused aromatic rings and two charges can assemble into stable thread-like noncovalent polymers that may further form liquid crystal phases in water, traditionally termed chromonic liquid crystals. Using five of these mesogenic molecules as additives to induce protein crystallization, we discover that molecules that can form liquid crystal phases in water are highly effective at inducing the crystal formation of lysozyme, even at concentrations significantly lower than that required for forming liquid crystal phases. This result reveals an example of inducing protein crystallization by the molecular assembly of the additives, and is consistent with a new mechanism by which the strong hydration of an assembly process provides a gradual means to compete for the water molecules to enable solvated proteins to form crystals.