Rv0100: An essential acyl carrier protein from M. tuberculosis important in dormancy.

We have identified an acyl-carrier protein, Rv0100, that is up-regulated in a dormancy model. This protein plays a critical role in the fatty acid biosynthesis pathway, which is important for energy storage and cell wall synthesis in Mycobacterium tuberculosis (MTB). Knocking out the Rv0100 gene resulted in a significant reduction of growth compared to wild-type MTB in the Wayne model of non-replicating persistence. We have also shown that Rv0100 is essential for the growth and survival of this pathogen during infection in mice and a macrophage model. Furthermore, knocking out Rv0100 disrupted the synthesis of phthiocerol dimycocerosates, the virulence-enhancing lipids produced by MTB and Mycobacterium bovis. We hypothesize that this essential gene contributes to MTB virulence in the state of latent infection. Therefore, inhibitors targeting this gene could prove to be potent antibacterial agents against this pathogen.

Pharmacological inhibition of CLK2 activates YAP by promoting alternative splicing of AMOTL2.

Yes-associated protein (YAP), the downstream effector of the evolutionarily conserved Hippo pathway, promotes cellular proliferation and coordinates certain regenerative responses in mammals. Small molecule activators of YAP may, therefore, display therapeutic utility in treating disease states involving insufficient proliferative repair. From a high-throughput chemical screen of the comprehensive drug repurposing library ReFRAME, here we report the identification of SM04690, a clinical stage inhibitor of CLK2, as a potent activator of YAP-driven transcriptional activity in cells. CLK2 inhibition promotes alternative splicing of the Hippo pathway protein AMOTL2, producing an exon-skipped gene product that can no longer associate with membrane-bound proteins, resulting in decreased phosphorylation and membrane localization of YAP. This study reveals a novel mechanism by which pharmacological perturbation of alternative splicing inactivates the Hippo pathway and promotes YAP-dependent cellular growth.

Pharmacological YAP activation promotes regenerative repair of cutaneous wounds.

Chronic cutaneous wounds remain a persistent unmet medical need that decreases life expectancy and quality of life. Here, we report that topical application of PY-60, a small-molecule activator of the transcriptional coactivator Yes-associated protein (YAP), promotes regenerative repair of cutaneous wounds in pig and human models. Pharmacological YAP activation enacts a reversible pro-proliferative transcriptional program in keratinocytes and dermal cells that results in accelerated re-epithelization and regranulation of the wound bed. These results demonstrate that transient topical administration of a YAP activating agent may represent a generalizable therapeutic approach to treating cutaneous wounds.

Pharmacological inhibition of CLK2 activates YAP by promoting alternative splicing of AMOTL2.

Yes-associated protein (YAP), the downstream effector of the evolutionarily conserved Hippo pathway, promotes cellular proliferation and coordinates certain regenerative responses in mammals. Small molecule activators of YAP may therefore display therapeutic utility in treating disease states involving insufficient proliferative repair. From a high-throughput chemical screen of the comprehensive drug repurposing library ReFRAME, here we report the identification of SM04690, a clinical stage inhibitor of CLK2, as a potent activator of YAP driven transcriptional activity in cells. CLK2 inhibition promotes alternative splicing of the Hippo pathway protein AMOTL2, producing an exon-skipped gene product that can no longer associate with membrane-bound proteins, resulting in decreased phosphorylation and membrane localization of YAP. This study reveals a novel mechanism by which pharmacological perturbation of alternative splicing inactivates the Hippo pathway and promotes YAP dependent cellular growth.

YAP-dependent proliferation by a small molecule targeting annexin A2.

The transcriptional coactivator Yes-associated protein 1 (YAP) orchestrates a proproliferative transcriptional program that controls the fate of somatic stem cells and the regenerative responses of certain tissues. As such, agents that activate YAP may hold therapeutic potential in disease states exacerbated by insufficient proliferative repair. Here we report the discovery of a small molecule, termed PY-60, which robustly activates YAP transcriptional activity in vitro and promotes YAP-dependent expansion of epidermal keratinocytes in mouse following topical drug administration. Chemical proteomics revealed the relevant target of PY-60 to be annexin A2 (ANXA2), a protein that directly associates with YAP at the cell membrane in response to increased cell density. PY-60 treatment liberates ANXA2 from the membrane, ultimately promoting a phosphatase-bound, nonphosphorylated and transcriptionally active form of YAP. This work reveals ANXA2 as a previously undescribed, druggable component of the Hippo pathway and suggests a mechanistic rationale to promote regenerative repair in disease.

Strategies in anti-Mycobacterium tuberculosis drug discovery based on phenotypic screening.

The rise of multi- and extensively drug-resistant Mycobacterium tuberculosis (M. tb) strains and co-infection with human immunodeficiency virus has escalated the need for new anti-M. tb drugs. Numerous challenges associated with the M. tb, in particular slow growth and pathogenicity level 3, discouraged use of this organism in past primary screening efforts. From current knowledge of the physiology and drug susceptibility of mycobacteria in general and M. tb specifically, it can be assumed that many potentially useful drug leads were missed by failing to screen directly against this pathogen. This review discusses recent high-throughput phenotypic screening strategies for anti-M. tb drug discovery. Emphasis is placed on prioritization of hits, including their extensive biological and chemical profiling, as well as the development status of promising drug candidates discovered with phenotypic screening.

Rufomycin Targets ClpC1 Proteolysis in Mycobacterium tuberculosis and M. abscessus.

ClpC1 is an emerging new target for the treatment of Mycobacterium tuberculosis infections, and several cyclic peptides (ecumicin, cyclomarin A, and lassomycin) are known to act on this target. This study identified another group of peptides, the rufomycins (RUFs), as bactericidal to M. tuberculosis through the inhibition of ClpC1 and subsequent modulation of protein degradation of intracellular proteins. Rufomycin I (RUFI) was found to be a potent and selective lead compound for both M. tuberculosis (MIC, 0.02 µM) and Mycobacterium abscessus (MIC, 0.4 µM). Spontaneously generated mutants resistant to RUFI involved seven unique single nucleotide polymorphism (SNP) mutations at three distinct codons within the N-terminal domain of clpC1 (V13, H77, and F80). RUFI also significantly decreased the proteolytic capabilities of the ClpC1/P1/P2 complex to degrade casein, while having no significant effect on the ATPase activity of ClpC1. This represents a marked difference from ecumicin, which inhibits ClpC1 proteolysis but stimulates the ATPase activity, thereby providing evidence that although these peptides share ClpC1 as a macromolecular target, their downstream effects are distinct, likely due to differences in binding.

QSAR-driven design, synthesis and discovery of potent chalcone derivatives with antitubercular activity.

New anti-tuberculosis (anti-TB) drugs are urgently needed to battle drug-resistant Mycobacterium tuberculosis strains and to shorten the current 6-12-month treatment regimen. In this work, we have continued the efforts to develop chalcone-based anti-TB compounds by using an in silico design and QSAR-driven approach. Initially, we developed SAR rules and binary QSAR models using literature data for targeted design of new heteroaryl chalcone compounds with anti-TB activity. Using these models, we prioritized 33 compounds for synthesis and biological evaluation. As a result, 10 heteroaryl chalcone compounds (4, 8, 9, 11, 13, 17-20, and 23) were found to exhibit nanomolar activity against replicating mycobacteria, low micromolar activity against nonreplicating bacteria, and nanomolar and micromolar against rifampin (RMP) and isoniazid (INH) monoresistant strains (rRMP and rINH) (<1 µM and <10 µM, respectively). The series also show low activity against commensal bacteria and generally show good selectivity toward M. tuberculosis, with very low cytotoxicity against Vero cells (SI = 11-545). Our results suggest that our designed heteroaryl chalcone compounds, due to their high potency and selectivity, are promising anti-TB agents.

Sweet spot matching: A thin-layer chromatography-based countercurrent solvent system selection strategy.

TLC-based strategies were proposed in 1979 (Hostettmann et al.) and 2005 (Friesen & Pauli; GUESS method) to minimize the number of partitioning experiments required for countercurrent separation (CCS) solvent system selection. As semi-empirical approaches, both proposed that the K values defining the sweet spot of optimal CCS corresponded to a matching Rf value range from the silica gel TLC plate developed in the organic phase of a biphasic or a corresponding monophasic solvent system. Despite their simplicity, there has been an absence of theoretical support and a deficiency of reported experimental evidence. The present study explores the theory required to develop correlations between Rf and K. All theoretical models surmise that the optimal Rf value range should be centered at 0.5. In order to validate the feasibility of the concept of matching Rf and K values, 43 natural products and six solvent system families were investigated. Out of 62 correlations, 45 resulted in matched Rf and K values. Based on this study, practical guidelines for the TLC-based prediction strategy are provided. These approaches will equip CCS users with an updated understanding of how to apply the TLC-based solvent system selection strategy to accelerate a targeted selection of CCS conditions.

Bioautography with TLC-MS/NMR for Rapid Discovery of Anti-tuberculosis Lead Compounds from Natural Sources.

While natural products constitute an established source of lead compounds, the classical iterative bioassay-guided isolation process is both time- and labor-intensive and prone to failing to identify active minor constituents. (HP)TLC-bioautography-MS/NMR, which combines cutting-edge microbiological, chromatographic, and spectrometric technologies, was developed to accelerate anti-tuberculosis (TB) drug discovery from natural sources by acquiring structural information at a very early stage of the isolation process. Using the avirulent, bioluminescent Mtb strain mc27000 luxABCDE, three variations of bioautography were evaluated and optimized for sensitivity in detecting anti-TB agents, including established clinical agents and new leads with novel mechanisms of action. Several exemplary applications of this approach to microbial extracts demonstrate its potential as a routine method in anti-TB drug discovery from natural sources.

Development of a novel direct bioautography-thin-layer chromatography test: optimization of growth conditions for gram-positive bacteria, Bacillus subtilis.

A TLC-direct bioautography (DB) assay using Bacillus subtilis as test bacteria was developed. Various factors affecting the microorganism's viability on the TLC plates were studied and verified for the flumequine standards. The Dhenasar's method called "direct sample determination" was used for TLC; the antibiotic samples were spotted on the TLC plates and subjected to bioautography without developing with a mobile phase. The best preincubation and incubation times of bacterial broth were found to be 1 h at 37 degrees C and 6 h at 37 degrees C. The optimal viscosity of broth was obtained by the addition of agarose to obtain a 0.05% solution in the Mueller-Hinton broth. The best incubation time of seeded TLC plates was 17 h at 37 degrees C. The plates were visualized by spraying with 0.2% aqueous 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide solution and incubated again for 0.5 h at 37 degrees C. The method was validated by determination of linearity, interday and intraday precision, LOD, and LOQ. The calibration curves showed good linearity in the range 0.005-0.5 microg (0.5-50.0 microg/mL). The regression coefficients were 0.9970 and 0.9955 for intraday and interday plots, respectively. The LOD of flumequine equalled 0.5 microg/mL, i.e., 5 ng of the antibiotic in the spot. The sensitivity of the developed TLC-DB test was compared with that of the two most commonly used standard antimicrobial susceptibility assays: agar disc diffusion and agar cylinder diffusion. The obtained minimum inhibitory concentration values clearly indicate much higher sensitivity of the TLC-DB method compared to the standard antimicrobial susceptibility assays.

Applications of novel direct bioautography tests for analysis of antimicrobials: a review.

TLC coupled to direct bioautography detection can be applied to the analysis of various antimicrobial agents in complex matrixes. Because of the lack of commercially available microbiological detection methods, two direct bioautography tests were developed in our laboratory to be used after TLC separation. One method was based on Gram-positive bacteria, Bacillus subtilis, and the other on Gram-negative bacteria, Escherichia coli. These tests can be used for detection and determination of wide spectrum of antimicrobials as well as for other, nontypical purposes, such as choosing the best sample preparation method before the analysis. Some of the more interesting applications of the newly developed tests are described in this paper.

Comparison of deproteinization methods used before TLC-DB and HPLC analysis of flumequine residues in milk.

Seventeen various extraction procedures based on precipitation of proteins in milk samples spiked with flumequine were tested. Several criteria were taken into account, when choosing the most effective. The supernatants were analyzed by thin-layer chromatography direct bioautography (TLC-DB) and high performance liquid chromatography (HPLC-UV). The results obtained from both methods indicate as the best the same deproteinization procedure. The addition of acetonitrile to milk in 1:1 volume proportions gave the highest concentration of flumequine in supernatant and prompt coagulation of proteins in milk samples.

Development of a novel direct bioautography-thin-layer chromatography test: optimization of growth conditions for gram-negative bacteria, Escherichia coli.

With the aim of developing a TLC-direct bioautography assay using Escherichia coli as test bacteria, various parameters influencing the viability of microorganisms on TLC plates were examined and checked for flumequine standards. The optimal times for preincubation and incubation of bacterial broth were 20 h at 37 degrees C and 2 h at 37 degrees C, respectively. The optimal viscosity of the broth was obtained for 0.05% agarose solution in Mueller-Hinton broth. Various incubation times of the seeded TLC plates were also tested (5 h proved to be optimal). After incubation, the plates were sprayed with 0.2% aqueous [3-(4,5-dimethyldiazol-2-yl)-2,5 diphenyltetrazolium bromide] (MTT) solution and incubated for 0.5 h at 37 degrees C. The precision of the method was evaluated by the repeatability (intraday assay) and intermediate precision (interday assay). The regression coefficients were 0.9977 and 0.9968, respectively, for intraday and interday curves. The calibration curves show good linearity in the range of 0.005-0.50 microg (0.5-50.0 microg/mL). The established LOD of flumequine equaled 0.5 microg/mL, i.e., 5 ng flumequine in the spot. The developed direct bioautography test significantly enhances the sensitivity of the TLC method.

Bioautography detection in thin-layer chromatography.

Bioautography is a microbial detection method hyphenated with planar chromatography techniques. It is based mainly on antimicrobial or antifungal properties of analyzed substances. The review discusses three versions of bioautography, i.e. contact, immersion and direct bioautography. The more concern is given to the last one. Many applications are quoted, not only for testing various groups of compounds, but also for investigating biochemical processes and factors influencing bacterial growth. Additionally, related methods, which can be included into direct bioautography, are discussed. The most promising among them seems to be TLC-bioluminescence screening.