Vibha Pathak

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.

Atomic structures of human dihydrofolate reductase complexed with NADPH and two lipophilic antifolates at 1.09 a and 1.05 a resolution.

The crystal structures of two human dihydrofolate reductase (hDHFR) ternary complexes, each with bound NADPH cofactor and a lipophilic antifolate inhibitor, have been determined at atomic resolution. The potent inhibitors 6-([5-quinolylamino]methyl)-2,4-diamino-5-methylpyrido[2,3-d]pyrimidine (SRI-9439) and (Z)-6-(2-[2,5-dimethoxyphenyl]ethen-1-yl)-2,4-diamino-5-methylpyrido[2,3-d]pyrimidine (SRI-9662) were developed at Southern Research Institute against Toxoplasma gondii DHFR-thymidylate synthase. The 5-deazapteridine ring of each inhibitor adopts an unusual puckered conformation that enables the formation of identical contacts in the active site. Conversely, the quinoline and dimethoxybenzene moieties exhibit distinct binding characteristics that account for the differences in inhibitory activity. In both structures, a salt-bridge is formed between Arg70 in the active site and Glu44 from a symmetry-related molecule in the crystal lattice that mimics the binding of methotrexate to DHFR.

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.

Imidazolium Cation Supported Solution-Phase Assembly of Homolinear α(1→6)−Linked Octamannoside: An Efficient Alternate Approach for Oligosaccharide Synthesis

An efficient, simple convergent assembly of a homolinear alpha(1-->6)-linked octamannosyl thioglycoside was obtained starting from imidazolium cation-tagged mannosyl fluoride and thiomannoside using block couplings. During chain elongation glycosylation reactions no column chromatographic purifications were used.

A facile method for deprotection of trityl ethers using column chromatography

A mild, efficient and inexpensive detritylation method is reported that uses trifluoroacetic acid on a silica gel column to obtain pure, detritylated compounds in one-step. This method is applicable to acid stable as well as acid sensitive compounds with only slight alterations in the procedure. Nineteen examples are given.

Lipoarabinomannan biosynthesis in Corynebacterineae: the interplay of two α(1→2)-mannopyranosyltransferases MptC and MptD in mannan branching

Lipomannan (LM) and lipoarabinomannan (LAM) are key Corynebacterineae glycoconjugates that are integral components of the mycobacterial cell wall, and are potent immunomodulators during infection. LAM is a complex heteropolysaccharide synthesized by an array of essential glycosyltransferase family C (GT‐C) members, which represent potential drug targets. Herein, we have identified and characterized two open reading frames from Corynebacterium glutamicum that encode for putative GT‐Cs. Deletion of NCgl2100 and NCgl2097 in C. glutamicum demonstrated their role in the biosynthesis of the branching α(1→2)‐Manp residues found in LM and LAM. In addition, utilizing a chemically defined nonasaccharide acceptor, azidoethyl 6‐O‐benzyl‐α‐D‐mannopyranosyl‐(1→6)‐[α‐D‐mannopyranosyl‐(1→6)]7‐D‐mannopyranoside, and the glycosyl donor C50‐polyprenol‐phosphate‐[14C]‐mannose with membranes prepared from different C. glutamicum mutant strains, we have shown that both NCgl2100 and NCgl2097 encode for novel α(1→2)‐mannopyranosyltransferases, which we have termed MptC and MptD respectively. Complementation studies and in vitro assays also identified Rv2181 as a homologue of Cg‐MptC in Mycobacterium tuberculosis. Finally, we investigated the ability of LM and LAM from C. glutamicum, and C. glutamicumΔmptC and C. glutamicumΔmptD mutants, to activate Toll‐like receptor 2. Overall, our study enhances our understanding of complex lipoglycan biosynthesis in Corynebacterineae and sheds further light on the structural and functional relationship of these classes of polysaccharides.

Synthesis of Deoxygenated (α 1→5)-Linked Arabinofuranose Disaccharides as Substrates and Inhibitors of Arabinosyltransferases of Mycobacterium tuberculosis

Arabinosyltransferases (AraTs) play a critical role in mycobacterial cell wall biosynthesis and are potential drug targets for the treatment of tuberculosis, especially multi-drug resistant forms of M. tuberculosis (MTB). Herein, we report the synthesis and acceptor/inhibitory activity of Araf alpha(1-->5) Araf disaccharides possessing deoxygenation at the reducing sugar of the disaccharide. Deoxygenation at either the C-2 or C-3 position of Araf was achieved via a free radical procedure using xanthate derivatives of the hydroxyl group. The alpha(1-->5)-linked disaccharides were produced by coupling n-octyl alpha-Araf 2-/3-deoxy, 2-fluoro glycosyl acceptors with an Araf thioglycosyl donor. The target disaccharides were tested in a cell free mycobacterial AraTs assay as well as an in vitro assay against MTB H(37)Ra and M. avium complex strains.

Synthesis of a fluorescent arabinofuranosyl disaccharide: a probe for arabinosyltransferase activity in Mycobacterium tuberculosis

Abstract (5- N , N -Dimethylaminonaphthalene-1-sulfonamidoethyl)-5- O -(α- d -arabinofuranosyl)-α- d -arabinofuranoside 1 was synthesized as a fluorescent probe for the determination of arabinosyltransferase activity in Mycobacterium tuberculosis .