Fueling Open‐Source Drug Discovery: 177 Small‐Molecule Leads against Tuberculosis

With the aim of fuelling open‐source, translational, early‐stage drug discovery activities, the results of the recently completed antimycobacterial phenotypic screening campaign against Mycobacterium bovis BCG with hit confirmation in M. tuberculosis H37Rv were made publicly accessible. A set of 177 potent non‐cytotoxic H37Rv hits was identified and will be made available to maximize the potential impact of the compounds toward a chemical genetics/proteomics exercise, while at the same time providing a plethora of potential starting points for new synthetic lead‐generation activities. Two additional drug‐discovery‐relevant datasets are included: a) a drug‐like property analysis reflecting the latest lead‐like guidelines and b) an early lead‐generation package of the most promising hits within the clusters identified.     

New Investigations with Lupane Type A-Ring Azepane Triterpenoids for Antimycobacterial Drug Candidate Design

Twenty lupane type A-ring azepano-triterpenoids were synthesized from betulin and its related derivatives and their antitubercular activity against Mycobacterium tuberculosis, mono-resistant MTB strains, and nontuberculous strains Mycobacterium abscessus and Mycobacterium avium were investigated in the framework of AToMIc (Anti-mycobacterial Target or Mechanism Identification Contract) realized by the Division of Microbiology and Infectious Diseases, NIAID, National Institute of Health. Of all the tested triterpenoids, 17 compounds showed antitubercular activity and 6 compounds were highly active on the H37Rv wild strain (with MIC 0.5 µM for compound 7), out of which 4 derivatives also emerged as highly active compounds on the three mono-resistant MTB strains. Molecular docking corroborated with a machine learning drug-drug similarity algorithm revealed that azepano-triterpenoids have a rifampicin-like antitubercular activity, with compound 7 scoring the highest as a potential M. tuberculosis RNAP potential inhibitor. FIC testing demonstrated an additive effect of compound 7 when combined with rifampin, isoniazid and ethambutol. Most compounds were highly active against M. avium with compound 14 recording the same MIC value as the control rifampicin (0.0625 µM). The antitubercular ex vivo effectiveness of the tested compounds on THP-1 infected macrophages is correlated with their increased cell permeability. The tested triterpenoids also exhibit low cytotoxicity and do not induce antibacterial resistance in MTB strains.     

Identification of a New Diterpene Biosynthetic Gene Cluster that Produces O‐Methylkolavelool in Herpetosiphon aurantiacus

Diterpenoids are usually found in plants and fungi, but are rare in bacteria. We have previously reported new diterpenes, named tuberculosinol and isotuberculosinol, which are generated from the Mycobacterium tuberculosis gene products Rv3377c and Rv3378c. No homologous gene was found at that time, but we recently found highly homologous proteins in the Herpetosiphon aurantiacus ATCC 23779 genome. Haur_2145 was a class II diterpene cyclase responsible for the conversion of geranylgeranyl diphosphate into kolavenyl diphosphate. Haur_2146, homologous to Rv3378c, synthesized (+)‐kolavelool through the nucleophilic addition of a water molecule to the incipient cation formed after the diphosphate moiety was released. Haur_2147 afforded (+)‐O‐methylkolavelool from (+)‐kolavelool, so this enzyme was an O‐methyltransferase. This new diterpene was indeed detected in H. aurantiacus cells. This is the first report of the identification of a (+)‐O‐methylkolavelool biosynthetic gene cluster.     

Mycobacterium tuberculosis Rv1916 is an Acetyl-CoA-Binding Protein

Isocitrate lyase (ICL) isoform 2 is an essential enzyme for some clinical Mycobacterium tuberculosis (Mtb) strains during infection. In the laboratory Mtb strain H37Rv, the icl2 gene encodes two distinct gene products – Rv1915 and Rv1916 – due to a frameshift mutation. This study aims to characterise these two gene products to understand their structure and function. While we were unable to produce Rv1915 recombinantly, soluble Rv1916 was obtained with sufficient yield for characterisation. Kinetic studies using UV-visible spectrophotometry and ¹H-NMR spectroscopy showed that recombinant Rv1916 does not possess isocitrate lyase activity, while waterLOGSY binding experiments demonstrated that it could bind acetyl-CoA. Finally, X-ray crystallography revealed structural similarities between Rv1916 and the C-terminal domain of ICL2. Considering the probable differences between full-length ICL2 and the gene products Rv1915 and Rv1916, care must be taken when using Mtb H37Rv as a model organism to study central carbon metabolism.     

Characterization of the Rv3377c Gene Product,a Type‐B Diterpene Cyclase,from the Mycobacterium tuberculosis H37 Genome

The Rv3377c gene from the Mycobacterium tuberculosis H37 genome is specifically limited to those Mycobacterium species that cause tuberculosis. We have demonstrated that the gene product of Rv3377c is a diterpene cyclase that catalyzes the formation of tuberculosinol from geranylgeranyl diphosphate (GGPP). However, the characteristics of this enzyme had not previously been studied in detail with homogeneously purified enzyme. The purified enzyme catalyzed the synthesis of tuberculosinyl diphosphate from GGPP, but it did not bring about the synthesis of tuberculosinol. Optimal conditions for the highest activity were found to be as follows: pH 7.5, 30 °C, Mg^II (0.1 mM ), and Triton X‐100 (0.1 %). Under these conditions, the kinetic values of K_M and k_cat were determined to be 11.7±1.9 μM for GGPP and 12.7±0.7 min^?1, respectively, whereas the specific activity was 186 nmol min^?1 mg^?1. The enzyme activity was inhibited at substrate concentrations higher than 50 μM . The catalytic activity was strongly inhibited by 15‐aza‐dihydrogeranylgeraniol and 5‐isopropyl‐N,N,N,2‐tetramethyl‐4‐(piperidine‐1‐carbonyloxy)benzenaminium chloride (Amo‐1618). The DXDTT_293–297 motif, corresponding to the DXDDTA motif conserved among terpene cyclases, was mutated in order to investigate its function. The middle D295 was found to be the most crucial entity for the catalysis. D293 and two threonine residues function synergistically to enhance the acidity of D295, possibly through hydrogen‐bonding networks. The Rv3377c enzyme could also react with (14R/S)‐14,15‐oxidoGGPP to generate 3α‐ and 3β‐hydroxytuberculosinyl diphosphate. Conformational analyses were carried out with deuterium‐labeled GGPP and oxidoGGPP. We found that GGPP and (14R)‐oxidoGGPP adopted a chair/chair conformation, but (14S)‐oxidoGGPP adopted a boat/chair conformation. Interestingly, the conformations of oxidoGGPP for the A‐ring formation are the opposite of those of oxidosqualene when it is used as a substrate by squalene cyclases for the biosynthesis of hopene and tetrahymanol. (3R)‐Oxidosqualene is folded in a boat conformation, whereas (3S)‐2,3‐oxidosqualene folds into a chair conformation, for the formation of the A‐rings of the hopene and tetrahymanol skeletons, respectively.     

Structure‐Guided Discovery of Antitubercular Agents That Target the Gyrase ATPase Domain

In this study we explored the pharmaceutically underexploited ATPase domain of DNA gyrase (GyrB) as a potential platform for developing novel agents that target Mycobacterium tuberculosis. In this effort a combination of ligand‐ and structure‐based pharmacophore modeling was used to identify structurally diverse small‐molecule inhibitors of the mycobacterial GyrB domain based on the crystal structure of the enzyme with a pyrrolamide inhibitor (PDB ID: 4BAE ). Pharmacophore modeling and subsequent in vitro screening resulted in an initial hit compound 5 [(E)‐5‐(5‐(2‐(1H‐benzo[d]imidazol‐2‐yl)‐2‐cyanovinyl)furan‐2‐yl)isophthalic acid; IC₅₀=4.6±0.1 μm ], which was subsequently tailored through a combination of molecular modeling and synthetic chemistry to yield the optimized lead compound 24 [(E)‐3‐(5‐(2‐cyano‐2‐(5‐methyl‐1H‐benzo[d]imidazol‐2‐yl)vinyl)thiophen‐2‐yl)benzoic acid; IC₅₀=0.3±0.2 μm ], which was found to display considerable in vitro efficacy against the purified GyrB enzyme and potency against the H₃₇Rv strain of M. tuberculosis. Structural handles were also identified that will provide a suitable foundation for further optimization of these potent analogues.     

2-Methoxylated fatty acids in marine sponges: Defense mechanism against mycobacteria?

A series of saturated 2-methoxylated FA having even-numbered chains with 8–14 carbons were synthesized, and their spectroscopic data are presented for the first time. The 2-methoxylated C₁₀−C₁₄ acids were prepared from the corresponding 2-hydroxylated FA, whereas the 2-methoxyoctanoic acid was synthesized starting with heptaldehyde. All of the methoxylated FA displayed some degree of inhibition (between 2 and 99%) of Mycobacterium tuberculosis H₃₇Rv at 6.25 μg/mL. The most inhibitory FA was 2-methoxydecanoic acid, with a minimum inhibitory concentration of 200–239 μM against M. tuberculosis H₃₇Rv as determined by both the microplate Alamar Blue assay and the green fluorescent protein microplate assay. These results are discussed in terms of the possible role of the 2-methoxylated FA as antimicrobial lipids produced either by marine sponges, or the associated marine symbiotic bacteria, as a defense mechanism in a highly competitive environment.     

Mycobacterium tuberculosis H37Rv Strain Increases the Frequency of CD3+TCR+ Macrophages and Affects Their Phenotype,but Not Their Migration Ability

In mycobacterial infections, the number of cells from two newly discovered subpopulations of CD3⁺ myeloid cells are increased at the infection site; one type expresses the T cell receptor (CD3⁺TCRαβ⁺) and the other does not (CD3⁺TCRαβ⁻). The role of Mycobacterium tuberculosis (Mtb) virulence in generating these subpopulations and the ability of these cells to migrate remains unclear. In this study, monocyte-derived macrophages (MDMs) infected in vitro with either a virulent (H37Rv) or an avirulent (H37Ra) Mtb strain were phenotypically characterized based on three MDM phenotypes (CD3⁻, CD3⁺TCRαβ⁺, and CD3⁺TCRαβ⁻); then, their migration ability upon Mtb infection was evaluated. We found no differences in the frequency of CD3⁺ MDMs at 24 h of infection with either Mtb strain. However, H37Rv infection increased the frequency of CD3⁺TCRαβ⁺ MDMs at a multiplicity of infection of 1 and altered the expression of CD1b, CD1c, and TNF on the surface of cells from both the CD3⁺ MDM subpopulations; it also modified the expression of CCR2, CXCR1, and CCR7, thus affecting CCL2 and IL-8 levels. Moreover, H37Rv infection decreased the migration ability of the CD3⁻ MDMs, but not CD3⁺ MDMs. These results confirm that the CD3⁺ macrophage subpopulations express chemokine receptors that respond to chemoattractants, facilitating cell migration. Together, these data suggest that CD3⁺ MDMs are a functional subpopulation involved in the immune response against Mtb.     

Gyrase ATPase Domain as an Antitubercular Drug Discovery Platform: Structure‐Based Design and Lead Optimization of Nitrothiazolyl Carboxamide Analogues

In this study, we explored the pharmaceutically underexploited mycobacterial gyrase ATPase (GyrB) domain as a template for a structure‐based virtual screening of our in‐house (BITS Pilani) compound collection to discover new inhibitors targeting Mycobacterium tuberculosis (M.tb.) The hit identified was further customized by using a combination of molecular docking and medicinal chemistry strategies to obtain an optimized analogue displaying considerable in vitro enzyme efficacy and bactericidal properties against the M.tb. H₃₇Rv strain. The binding affinity of the ligand toward the GyrB domain was reascertained by differential scanning fluorimetry experiments. Further evaluation of the hERG toxicity (a major limitation among the previously reported N‐linked aminopiperidine analogues) indicated these molecules to be completely devoid of cardiotoxicity, a significant achievement within this class.     

Preliminary Evaluation of Artemisinin–Cholesterol Conjugates as Potential Drugs for the Treatment of Intractable Forms of Malaria and Tuberculosis

To evaluate the feasibility of developing drugs that may be active against both malaria and tuberculosis (TB) by using in part putative cholesterol transporters in the causative pathogens and through enhancement of passive diffusion in granulomatous TB, artemisinin–cholesterol conjugates were synthesized by connecting the component molecules through various linkers. The compounds were screened in vitro against Plasmodium falciparum (Pf) and Mycobacterium tuberculosis (Mtb). Antimalarial activities (IC₅₀) against Pf drug‐sensitive NF54, and drug‐resistant K1 and W2 strains ranged from 0.03–2.6, 0.03–1.9, and 0.02–1.7 μm . Although the compounds are less active than the precursor artemisinin derivatives, the cholesterol moiety renders the compounds relatively insoluble in the culture medium, and variation in solubilities among the different compounds may reflect in the range of efficacies observed. Activities against Mtb H37Rv were assessed using a standardized colony‐forming unit (CFU) assay after 24 h pretreatment of cultures with each of the compounds. Percentage inhibition ranged from 3–38 % and 18–52 % at 10 and 80 μm , respectively. Thus, in contrast to the comparator drug artemether, the conjugates display enhanced activities. The immediate aims include the preparation of conjugates with enhanced aqueous solubilities, assays against malaria and TB in vivo, and for TB, assays using an infected macrophage model and assessment of granuloma influx.     

Triazolophthalazines: Easily Accessible Compounds with Potent Antitubercular Activity

Tuberculosis (TB) remains one of the major causes of death worldwide, in particular because of the emergence of multidrug‐resistant TB. Herein we explored the potential of an alternative class of molecules as anti‐TB agents. Thus, a series of novel 3‐substituted triazolophthalazines was quickly and easily prepared from commercial hydralazine hydrochloride as starting material and were further evaluated for their antimycobacterial activities and cytotoxicities. Four of the synthesized compounds were found to effectively inhibit the Mycobacterium tuberculosis (M.tb) H₃₇Rv strain with minimum inhibitory concentration (MIC) values <10 μg mL^?1, whereas no compounds displayed cytotoxicity against HCT116 human cell lines (IC₅₀>100 μm ). More remarkably, the most potent compounds proved to be active to a similar extent against various multidrug‐resistant M.tb strains, thus uncovering a mode of action distinct from that of standard antitubercular agents. Overall, their ease of preparation, combined with their attractive antimycobacterial activities, make such triazolophthalazine‐based derivatives promising leads for further development.     

Hydrazone-Tethered 5-(Pyridin-4-yl)-4H-1,2,4-triazole-3-thiol Hybrids: Synthesis,Characterisation, in silico ADME Studies,and in vitro Antimycobacterial Evaluation and Cytotoxicity

Compounds containing arylpyrrole-, 1,2,4-triazole- and hydrazone structural frameworks have been widely studied and demonstrated to exhibit a wide range of pharmacological properties. Herein, an exploratory series of new 1,2,4-triazole derivatives designed by amalgamation of arylpyrrole and 1,2,4-triazole structural units via a hydrazone linkage is reported. The synthesised compounds were tested in vitro for their potential activity against Mycobacterium tuberculosis (MTB) H₃₇Rv strain. The most promising compound 13 – the derivative without the benzene ring appended to the pyrrole unit displayed acceptable activity (MIC₉₀=3.99 μM) against MTB H₃₇Rv, while other compounds from the series exhibited modest to weak antimycobacterial activity with MIC₉₀ values in the range between 7.0 and >125 μM. Furthermore, in silico results, predicated using the SwissADME web tool, show that the prepared compounds display desirable ADME profile with parameters within acceptable range.     

Synthesis and characterization of high molecular weight metaloquinolate‐containing polymers

In this article, a new method to synthesize novel metaloquinolate (AlQ₃, ZnQ₂)‐containing polymers is reported. A model polymer with 8‐hydroxyquinoline ligands can be obtained by free‐radical copolymerization with methyl methacrylate (MMA), then metaloquinolate (AlQ₃, ZnQ₂)‐containing polymers are prepared by coordinating reaction with di(8‐hydroxyquinoline) aluminum (AlQ₂) chelates or mono (8‐hydroxyquinoline) zinc (ZnQ) chelates without crosslinking. The structures of products are confirmed by NMR, FTIR, ultraviolet‐visible, elementary analysis, photoluminescence spectrum, and gel permeation chromatography analysis. They are soluble in common solvents and suitable to form films. The use of AlQ₂ and ZnQ avoided the crosslinking caused by the AlQ₃, ZnQ₂ formation between different polymer chains. Different from the traditional small organic molecules in organic light‐emitting diodes (OLEDs) fabrication, the polymer can be processed by spin coating without phase separation. Compared to the PMMA or MMA‐co‐HEMA‐CH₂‐Hq, the T_g of the metaloquinolate‐containing polymers was much higher. It should be of interest for OLED applications. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci 99: 1945–1952, 2006     

Inhibition of Mycobacterium tuberculosis methionine aminopeptidases by bengamide derivatives

Methionine aminopeptidase (MetAP) carries out an essential function of protein N‐terminal processing in many bacteria and is a promising target for the development of novel antitubercular agents. Natural bengamides potently inhibit the proliferation of mammalian cells by targeting MetAP enzymes, and the X‐ray crystal structure of human type 2 MetAP in complex with a bengamide derivative reveals the key interactions at the active site. By preserving the interactions with the conserved residues inside the binding pocket while exploring the differences between bacterial and human MetAPs around the binding pocket, seven bengamide derivatives were synthesized and evaluated for inhibition of MtMetAP1a and MtMetAP1c in different metalloforms, inhibition of M. tuberculosis growth in replicating and non‐replicating states, and inhibition of human K562 cell growth. Potent inhibition of MtMetAP1a and MtMetAP1c and modest growth inhibition of M. tuberculosis were observed for some of these derivatives. Crystal structures of MtMetAP1c in complex with two of the derivatives provided valuable structural information for improvement of these inhibitors for potency and selectivity.     

Biodegradation of quinoline in crude oil

Removal of quinoline, which is typical of nitrogen‐containing compounds in crude oil, was achieved by a biodegradation reaction by Comamonas sp TKV3‐2‐1. The aerobic strain, Comamonas sp TKV3‐2‐1, which can grow utilizing quinoline as the sources of both carbon and nitrogen, degraded quinoline to 2‐hydroxyquinoline, finally to water‐soluble substances. The degradation reaction of 2‐hydroxyquinoline was revealed to be regarded as a rate‐limiting step controlling the overall reaction of biodenitrogenation process of quinoline in crude oil. The degradation rate of 2‐hydroxyquinoline in a stirred fermenter had a maximum of 211 mg 2‐hydroxyquinoline g‐cell^?1 h^?1 when the portion of crude oil in the reaction mixture, the cell concentration and the rotational speed of agitation impeller were 83.3%(v/v), 28.5 gdm^?3 and 11.7 s^?1, respectively. After the reaction was completed, the crude oil and the cell suspension could be separated efficiently by centrifuging. The possibility of constructing a bioprocess for removing quinoline in crude oil under storage is also discussed. © 2001 Society of Chemical Industry     

Synthetic resin. XII. Synthesis and characterization of new terpolymer resins derived from 8‐hydroxyquinoline 5‐sulfonic acid,melamine, and formaldehyde

Terpolymers 8‐hydroxyquinoline 5‐sulphonic acid–melamine–formaldehyde (8‐HQ5‐SAMF) were synthesized through the condensation of 8‐hydroxyquinoline 5‐sulfonic acid and melamine with formaldehyde in the presence of an acid catalyst. Four different terpolymers were synthesized with various molar proportions of the reacting monomers. The terpolymer resin compositions were determined on the basis of elemental analysis. The number‐average molecular weights of these resins were determined by conductometric titration in a nonaqueous medium; viscometric measurements in dimethyl sulfoxide were carried out to ascertain the characteristic functions and constants. Ultraviolet–visible, Fourier transform infrared, ¹H‐NMR, and ¹³C‐NMR spectroscopy and thermogravimetric analysis were used to elucidate the structures. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2010     

Synthesis,exchange reactions,and biological activity of poly(8‐methacryloxyquinoline)

8‐Methacryloxyquinoline (MAQ) was prepared through the reaction of 8‐hydroxyquinoline with either methacryloyl chloride or methacrylic acid in the presence of triethylamine or N,N‐dicyclohexylcarbodiimide, respectively. MAQ was polymerized in dimethylformamide with 2,2‐azobisisobutyronitrile as the initiator. The reactions of the resulting polymers with hydroxyl and amino compounds were studied. The polymers were characterized with IR, ¹H‐NMR, and mass spectroscopy. Some of the synthesized polymers were tested for their antimicrobial activity against bacteria and fungi. Generally, all the polymers were effective against the tested microorganisms, but their growth‐inhibition effects varied. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

Semisynthetic Ursolic Acid Fluorolactone Derivatives Inhibit Growth with Induction of p21(waf1) and Induce Apoptosis with Upregulation of NOXA and Downregulation of c-FLIP in Cancer Cells

A series of ursolic acid ((1S,2R,4aS,6aR,6aS,6bR,8aR,10S,12aR,14bS)‐10‐hydroxy‐1,2,6a,6b,9,9,12a‐heptamethyl‐2,3,4,5,6,6a,7,8,8a,10,11,12,13,14b‐tetradecahydro‐1H‐picene‐4a‐carboxylic acid) derivatives with a 12‐fluoro‐13,28β‐lactone moiety were synthesized using the electrophilic fluorination reagent Selectfluor. The antiproliferative effects of these novel compounds were evaluated in AsPC‐1 pancreatic cancer cells, and the structure–activity relationships (SARs) were evaluated. Of the compounds synthesized, ursolic acid derivatives carrying a heterocyclic ring, such as imidazole or methylimidazole, and cyanoenones were among the more potent inhibitors of AsPC‐1 pancreatic cancer cell growth. 2‐Cyano‐3‐oxo‐12α‐fluoro‐urs‐1‐en‐13,28β‐olide, compound 20 , was the most effective inhibitor with IC₅₀ values of 0.7, 0.9 and 1.8 μM in pancreatic cancer cell lines AsPC‐1, MIA PaCa‐2 and PANC‐1, respectively. This compound also exhibited better antiproliferative activities against breast (MCF7), prostate (PC‐3), hepatocellular (Hep G2) and lung (A549) cancer cell lines, with IC₅₀ values lower than 1 μM . The mechanism of action by which these compounds exert their biological effect was evaluated in AsPC‐1 cells using the most potent inhibitor synthesized, compound 20 . At 1 μM , the cell cycle arrested at the G1 phase with upregulation of p21^waf1. Apoptosis was induced at an inhibitor concentration of 8 μM with upregulation of NOXA and downregulation of c‐FLIP. These data indicate that fluorolactone derivatives of ursolic acid have improved antiproliferative activity, acting through arrest of the cell cycle and induction of apoptosis.     

Rhodium‐Catalyzed [2+2+2] Cycloadditions of Diynes with Morita–Baylis–Hillman Adducts: A Stereoselective Entry to Densely Functionalized Cyclohexadiene Scaffolds

A rhodium‐catalyzed asymmetric synthesis of 5,5‐disubstituted cyclohexa‐1,3‐dienes has been achieved by [2+2+2] cycloaddition reactions between diynes and Morita–Baylis–Hillman (M‐B‐H) adducts as unsaturated substrates. Products containing two adjacent chiral centres (quaternary and tertiary, respectively) were obtained with complete diastereoselectivity and high enantioselectivity (84–97%) through a kinetic resolution of the M‐B‐H adduct. Furthermore, these highly substituted cyclohexadienes reacted with dienophiles to afford the corresponding Diels–Alder cycloadducts in good yields.

     

Inhibitors of the salicylate synthase (MbtI) from Mycobacterium tuberculosis discovered by high-throughput screening

A simple steady‐state kinetic high‐throughput assay was developed for the salicylate synthase MbtI from Mycobacterium tuberculosis, which catalyzes the first committed step of mycobactin biosynthesis. The mycobactins are small‐molecule iron chelators produced by M. tuberculosis, and their biosynthesis has been identified as a promising target for the development of new antitubercular agents. The assay was miniaturized to a 384‐well plate format and high‐throughput screening was performed at the National Screening Laboratory for the Regional Centers of Excellence in Biodefense and Emerging Infectious Diseases (NSRB). Three classes of compounds were identified comprising the benzisothiazolones (class I), diarylsulfones (class II), and benzimidazole‐2‐thiones (class III). Each of these compound series was further pursued to investigate their biochemical mechanism and structure–activity relationships. Benzimidazole‐2‐thione 4 emerged as the most promising inhibitor owing to its potent reversible inhibition.