William J. Suling

High Throughput Screening for Inhibitors of Mycobacterium tuberculosis H37Rv

There is an urgent need for the discovery and development of new antitubercular agents that target new biochemical pathways and treat drug resistant forms of the disease. One approach to addressing this need is through high-throughput screening of medicinally relevant libraries against the whole bacterium in order to discover a variety of new, active scaffolds that will stimulate new biological research and drug discovery. Through the Tuberculosis Antimicrobial Acquisition and Coordinating Facility (www.taacf.org), a large, medicinally relevant chemical library was screened against M. tuberculosis strain H37Rv. The screening methods and a medicinal chemistry analysis of the results are reported herein.

Antimycobacterial Activities of 2,4-Diamino-5-Deazapteridine Derivatives and Effects on Mycobacterial Dihydrofolate Reductase

ABSTRACT Development of new antimycobacterial agents for Mycobacterium avium complex (MAC) infections is important particularly for persons coinfected with human immunodeficiency virus. The objectives of this study were to evaluate the in vitro activity of 2,4-diamino-5-methyl-5-deazapteridines (DMDPs) against MAC and to assess their activities against MAC dihydrofolate reductase recombinant enzyme (rDHFR). Seventy-seven DMDP derivatives were evaluated initially for in vitro activity against one to three strains of MAC (NJ168, NJ211, and/or NJ3404). MICs were determined with 10-fold dilutions of drug and a colorimetric (Alamar Blue) microdilution broth assay. MAC rDHFR 50% inhibitory concentrations versus those of human rDHFR were also determined. Substitutions at position 5 of the pteridine moiety included -CH3, -CH2CH3, and -CH2OCH3 groups. Additionally, different substituted and unsubstituted aryl groups were linked at position 6 through a two-atom bridge of either -CH2NH, -CH2N(CH3), -CH2CH2, or -CH2S. All but 4 of the 77 derivatives were active against MAC NJ168 at concentrations of ≤13 μg/ml. Depending on the MAC strain used, 81 to 87% had MICs of ≤1.3 μg/ml. Twenty-one derivatives were >100-fold more active against MAC rDHFR than against human rDHFR. In general, selectivity was dependent on the composition of the two-atom bridge at position 6 and the attached aryl group with substitutions at the 2′ and 5′ positions on the phenyl ring. Using this assessment, a rational synthetic approach was implemented that resulted in a DMDP derivative that had significant intracellular activity against a MAC-infected Mono Mac 6 monocytic cell line. These results demonstrate that it is possible to synthesize pteridine derivatives that have selective activity against MAC.

Studies on (β,1→5) and (β,1→6) linked octyl Galf disaccharides as substrates for mycobacterial galactosyltransferase activity

Abstract The emergence of multi-drug resistant (MDR) strains of Mycobacterium tuberculosis (MTB) and the continuing pandemic of tuberculosis emphasizes the urgent need for the development of new anti-tubercular agents with novel drug targets. The recent structural elucidation of the mycobacterial cell wall highlights a large variety of structurally unique components that may be a basis for new drug development. This publication describes the synthesis, characterization, and screening of several octyl Gal f (β,1→5)Gal f and octyl Gal f (β,1→6)Gal f derivatives. A cell-free assay system has been utilized for galactosyltransferase activity using UDP[ 14 C]Gal f as the glycosyl donor, and in vitro inhibitory activity has been determined in a colorimetric broth microdilution assay system against MTB H37Ra and three clinical isolates of Mycobacterium avium complex (MAC). Certain derivatives showed moderate activities against MTB and MAC. The biological evaluation of these disaccharides suggests that more hydrophobic analogues with a blocked reducing end showed better activity as compared to totally deprotected disaccharides that more closely resemble the natural substrates in cell wall biosynthesis.

ETHAMBUTOL-SUGAR HYBRIDS AS POTENTIAL INHIBITORS OF MYCOBACTERIAL CELL-WALL BIOSYNTHESIS

Ethambutol is an established front-line agent for the treatment of tuberculosis, and is also active against Mycobacterium avium infection. However, this agent exhibits toxicity, and is considered to have low potency. The action of ethambutol on the mycobacterial cell wall, particularly the arabinan, and comparison of the structure of ethambutol with several of the cell-wall saccharides, suggested that ethambutol-saccharide hybrids might lead to agents with a more selective mechanism of action. To this end, eight ethambutol-saccharide hybrids were synthesized and screened against M. tuberculosis and several clinical isolates of M. avium.

Studies on n-Octyl-5-(α-d-arabinofuranosyl)-β-d-galactofuranosides for Mycobacterial Glycosyltransferase Activity

Abstract The mycobacterial cell wall is a potential target for new drug development. Herein we report the preparation and activity of several n -octyl-5-(α- d -arabinofuranosyl)-β- d -galactofuranoside derivatives. A cell-free assay system has been utilized for determination of the ability of disaccharide analogues to act as arabinosyltransferase acceptors using [ 14 C]-DPA as the glycosyl donor. In addition, in vitro inhibitory activity has been determined in a colorimetric broth microdilution assay system against MTB H37Ra and three clinical isolates of Mycobacterium avium complex (MAC). One of these disaccharides showed moderate activity against MTB. The biological evaluation of these disaccharides suggests that more hydrophobic analogues with a blocked reducing end showed better activity as compared to a totally deprotected disaccharide that more closely resembles the natural substrates in cell wall biosynthesis.

Studies on α(1→5) linked octyl arabinofuranosyl disaccharides for mycobacterial arabinosyl transferase activity

The appearance multi-drug resistant Mycobacterium tuberculosis (MTB) throughout the world has prompted a search for new, safer and more active agents against tuberculosis. Based on studies of the biosynthesis of mycobacterial cell wall polysaccharides, octyl 5-O-(alpha-D-arabinofuranosyl)-alpha-D-arabinofuranoside analogues were synthesized and evaluated as inhibitors for M. tuberculosis and Mycobacterium avium. A cell free assay system has been used for the evaluation of these disaccharides as substrates for mycobacterial arabinosyltransferase activity.

Homologated aza analogs of arabinose as antimycobacterial agents

A series of hydrolytically-stable aza analogs of arabinofuranose was prepared and evaluated against Mycobacterium tuberculosis and M. avium. The compounds were designed to mimic the putative arabinose donor involved in biogenesis of the essential cell wall polysaccharide, arabinogalactan. Though most compounds displayed little activity in cell culture, one compound showed significant activity in infected macrophage models.

Novel pyridopyrazine and pyrimidothiazine derivatives as FtsZ inhibitors.

A series of pyridopyrazine and pyrimidothiazine derivatives have been synthesized and their activity against FtsZ from Mycobacterium tuberculosis (Mtb) and in vitro antibacterial activity against Mtb H(37)Ra and Mtb H(37)Rv are reported. Certain analogs described herein showed moderate to good inhibitory activity.

Studies on β-d-Galf -(1→4)-α-l-Rhap octyl analogues as substrates for mycobacterial galactosyl transferase activity

Abstract The biochemically unique structures of sugar residues in the outer cell wall of Mycobacterium tuberculosis (MTB) make the pathways for their biosynthesis and utilization attractive targets for the development of new and selective anti-tubercular agents. A cell-free assay system for galactosyltransferase activity using UDP[ 14 C]Gal as the glycosyl donor, as well as an in vitro colorimetric broth micro-dilution assay system, were used to determine the activities of three β- d -gal f (1→4)-α- l -rham p octyl disaccharides as substrates and antimycobacterial agents respectively. The cell-free enzymatic studies using compounds 8 Download high-res image (126KB) Download full-size image Scheme 1 . Synthesis of disaccharides. Reagents and conditions: (a) Ac 2 O, Pyridine, rt, 4 h, 98%; (b) SnCl 4 , CH 3 (CH 2 ) 7 OH, CH 3 CN, 0°C–rt, 83%; (c) NaOMe, MeOH, rt, 2 h, 99%; (d) 2,2′-dimethoxypropane, (1S)-(+)-10-camphorsulphonic acid, acetone, rt, 2 h, 96%; (e) Hg(CN) 2 , HgBr 2 , CH 3 CN, 2 h, 79%; (f) NaOMe, MeOH, rt, 1 h, 90%; (g) TFA containing 1% H 2 O, CHCl 3 , rt, 3 h, 91%; (h) TFA containg 1% H 2 O, CHCl 3 rt, 3 h, 85%; (i) NaOMe, MeOH, rt, 1 h, 99%. and 10 suggested that these disaccharides bind to and are effective substrates for a putative mycobacterial galactosyltransferase. The modified acceptor 8 was found to be a slower but prolonged binder as compared to the less substituted analogue 10 as evidenced by their K m and V max values. Moderate antimycobacterial activity was observed with compounds 8 and 9 against MTB H37Ra and three clinical isolates of Mycobacterium avium complex (MAC).

The metabolism of 2-methyladenosine in Mycobacterium smegmatis

2-Methyladenosine (methyl-ado) has demonstrated selective activity against Mycobacterium tuberculosis, which indicates that differences in the substrate preferences between mycobacterial and human purine metabolic enzymes can be exploited to develop novel drugs for the treatment of mycobacterial diseases. Therefore, in an effort to better understand the reasons for the anti-mycobacterial activity of methyl-ado, its metabolism has been characterized in Mycobacterium smegmatis. In a wild-type strain, methyl-ado was phosphorylated by adenosine kinase to methyl-AMP, which was further converted to methyl-ATP and incorporated into RNA. In contrast, a mutant strain of M. smegmatis was isolated that was resistant to methyl-ado, deficient in adenosine kinase activity and was not able to generate methyl-ado metabolites in cells treated with methyl-ado. These results indicated that phosphorylated metabolites of methyl-ado were responsible for the cytotoxic activity of this compound. Methyl-ado was not a substrate for either adenosine deaminase or purine-nucleoside phosphorylase from M. smegmatis. Treatment of M. smegmatis with methyl-ado resulted in the inhibition of ATP synthesis, which indicated that a metabolite of methyl-ado inhibited one of the enzymes involved in de novo purine synthesis. These studies demonstrated the importance of adenosine kinase in the activation of methyl-ado to toxic metabolites in M. smegmatis.