Rational Design of New Antituberculosis Agents: Receptor-Independent Four-Dimensional Quantitative Structure−Activity Relationship Analysis of a Set of Isoniazid Derivatives

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  Rational Design of New Antituberculosis Agents: Receptor-Independent Four-Dimensional Quantitative Structure−Activity Relationship Analysis of a Set of Isoniazid Derivatives
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  Current Drug Targets, 2001  , 2,  427-437427  1389-4501/01 $28.00+.00© 2001 Bentham Science Publishers Ltd. An Approach for the Rational Design of New Antituberculosis Agents K.F.M. Pasqualoto *  and E.I. Ferreira Faculty of Pharmaceutical Sciences, University of São Paulo, Av Prof Lineu Prestes 580, 05508-900, São Paulo -SP, Brazil Abstract: Tuberculosis (TB) kills more youth and adults than any other infectious disease in the world today.The emergence of new strains of  Mycobacterium tuberculosis  resistant to some or all current antituberculosisdrugs is a serious and crescent problem. The resistance is often a corollary to HIV infection and drug-resistantTB is more difficult and more expensive to treat, besides to be more likely fatal. Thus, it is still necessary tosearch for new antimycobacterial agents. The identification of novel targets need the identification of  biochemical pathways specific to mycobacteria and related organisms. Many unique metabolic processes occur during the biosynthesis of mycobacterial cell wall components. In this report, we examine one of theseattractive targets for the rational design of new antituberculosis agents –the mycolic acids. THE DISEASE AND EPIDEMIOLOGY Tuberculosis (TB) is a chronic infectious disease caused  by mycobacteria of the “tuberculosis complex”, including  Mycobacterium bovis ,  Mycobacterium africanum  and mainly  Mycobacterium tuberculosis . TB is the classic example of adisease caused by an intracellular parasite. The infection isusually transmitted from person to person by the inhalationof infective droplet nuclei that result from the aerosolizationof respiratory secretions and may involve any organ system, but the lung is the usual site of the primary lesion and themain organ infected. The structure and evolution of lesionscaused by tubercle bacilli are determined by a host-specificdefense system, by immunological response and by geneticfactors [1,2]. According to the definition of the AmericanThoracic Society, TB infection does not mean disease. Thestate of disease is defined by the appearance of clinically,radiologically, and bacteriologically documentable signs and symptons of infection [1].According to World Health Organization (WHO), TBkills more youth and adults than any other infectious diseasein the world today. It is a worse killer than malaria and AIDS combined and kills more women than all the causes of maternal mortality. It is responsible for 100,000 childrendeaths each year. The global and regional incidence is nearly2 million TB cases per year in sub-Saharan Africa, nearly 3million TB cases per year in south-east Asia, over a quarter of a million TB cases per year in Eastern Europe and nearly161,800 new TB cases annually in Brazil. It is estimated that from now to 2020, nearly one billion more people will be newly infected, 200 million people will get sick, and 70million will die from tuberculosis –if control is notstrengthened [3,4].The pandemic of AIDS has had a major impact on theworldwide TB problem. One third of the increase in the *Address correspondence to this author at the Faculty of PharmaceuticalSciences, University of São Paulo, Av Prof Lineu Prestes 580, 05508-900,São Paulo - SP, Brazil; E-mail: kerly@netpoint.com.br  incidence of TB in the last five years can be attributed to co-infection with HIV. HIV weakens the immune system and the likelihood to become sick is increased in about 30 timesfor HIV-positive people and co-infected with TB than for those infected with TB who is HIV-negative [3]. Diagnosisis difficult because the characteristic pulmonary symptons,signs, and radiographic appearance often are absent [2]. WHOestimates that by the end of the century HIV infection willannually cause nearly 1.5 million cases of TB that otherwisewould not have occurred [3]. In Brazil, an estimated 200,000 people are believed to be co-infected with TB and HIV. This pattern is markedly high in Rio de Janeiro and São Paulo – cities with very high levels of both TB and HIV/AIDS [4].Other factor contributing to the rise in TB and responsible for the increased death rate is the emergence of new strains of  M. tuberculosis  resistant to some or allcurrent antituberculosis drugs, so called multidrug-resistantTB (MDR-TB). Up to 50 million people may be infected with drug-resistant TB. This resistance is caused byinconsistent or partial treatment, when patients do not havecompliance with long-term chemotherapy, the doctors and health workers prescribe the wrong drugs or the wrongcombination of drugs, or the drug supply is unreliable [3,5].The resistance is often a corollary to HIV infection [5]. Drug-resistant TB is more difficult and more expensive to treat,and more likely to be fatal. In industrialized countries TBtreatment costs around US $ 2,000 per patient, but risesfrom more than 100-fold up to US $ 250,000 per patientwith MDR-TB [3]. MDR-TB will be important to determinewhether the different mutations that can generate drugresistance differ in their effect on bacterial virulence.Different routes to development of resistance, such as katG mutation versus inha  mutation in the case of isoniazid, for example, might have quite different consequences in relationto their effect on the overall physiology and virulence of theresistant strains [6].Short-course chemotherapy forms the backbone of antituberculosis chemotherapy and this strategy of treatmentis recommended by WHO through the DOTS (DirectlyObserved Treatment, Short-course) to guarantee an effective  428   Current Drug Targets  , 2001  , Vol. 2, No. 4Pasqualoto and Ferreira TB control. Isoniazid (INH), rifampin (RIF), pyrazinamide(PZA), streptomycin (SM) and ethambutol (EMB) are thedrugs that constitute the antituberculosis short-coursechemotherapy. These drugs are typically administered for 6or 8 months in accordance with WHO’s TB treatmentguidelines [7]. SM has been available in the antituberculosischemotherapy since 1940s, INH and PZA since 1950s, EMBsince 1960s and RIF since 1970s [1,8]. DOTS combines fiveelements: political commitment, microscopy services, drugsupplies, monitoring systems and direct observation of treatment. The DOTS strategy prevents new infections and the development of MDR-TB. Unfortunately, DOTS is notwidely used yet. According to WHO, only 12 percent of estimated TB patients received DOTS in 1996; at the beginning of 1997, 95 out 212 countries had adopted theDOTS strategy and of those, 63 have implemented DOTScountrywide [3,7]. In Brazil, the DOTS strategy are now being concentrated in 250 municipalities selected for highincidence of TB, high mortality, low cure rate and spread of AIDS. The challenge is to spread DOTS more widely, assoon as possible, without risking the loss of quality whichcould create an incurable plague of MDR-TB [4].Although some general guidelines for the therapy of TBare accepted everywhere, great effort must be made to adaptthese guidelines to different socio-economical situations and to pathological variants. It is still necessary to search for newantimycobacterial agents for many reasons: drug resistance,serious side effects of some current drugs, and the lack of efficiency of current treatment in immunodepressed patients[1]. In addition to the investigation of resistance mechanismsfor individual drugs, an improved understanding of the basisof drug permeability of  Mycobacterium tuberculosis  is animportant goal for future research [6]. The permeability of mycobacteria to substances in their enviroment is controlled  by the properties of their envelopes. Two special features areimportant: an outer lipid barrier based on a monolayer of characteristic mycolic acids and a capsule-like coat of  polysaccharide and protein. The proper knowledge of mycobacterial permeability will enable new approaches to thetreatment of mycobacterial disease [9].Strategies based upon optimizing the inhibition of known targets require an extensive knowledge of the detailed mechanism of action of current antimycobacterial agents.Strategies based on the identification of novel targets willnecessitate the identification of biochemical pathwaysspecific to mycobacteria and related organisms. Many uniquemetabolic processes occur during the biosynthesis of mycobacterial cell wall components, and some attractive newtargets have emerged [10]. In this report, we examine one of these attractive targets for the rational design of newantituberculosis agents –the mycolic acids. MYCOBACTERIAL CELL WALL AND MYCOLICACID BIOSYNTHESIS Mycobacteria are a group of eubacteria that belong to alarger group of Gram-positive bacteria containing GC-richDNA, sometimes called the actinomycete line. Within thisgroup, mycobacteria belong to one branch, often called the Corynebacterium-Mycobacterium-Nocardia (CMN) branch,that produce cell walls of a unique structure, named chemotype IV cell wall. The cell walls of the usual Gram- positive bacteria are largely composed of peptidoglycan, withsome additional polysaccharides or polyol phosphate polymers (teichoic acids). The chemotype IV cell wallcontain meso -diaminopimelic acid as the diamino acid in the peptidoglycan, and, in contrast to the  N  -acetylation found inall other bacteria, the muramic acid residue is  N  -glycolylated in  Mycobacterium and   Nocardia . An important feature of thechemotype IV cell wall is the presence of a unique polysacharide, arabinogalactan, which is substituted by char-acteristic long-chain fatty acids, mycolic acids [11] (Fig. 1 ).Mycolic acids are high molecular-weight α -alkyl, β -hydroxy fatty acids, covalently linked to arabinogalactan. In Corynebacterium and  Nocardia , mycolic acids contain up toabout 40 and 60 carbon atoms, respectively, but those from  Mycobacterium  usually contain 70-90 carbons. Differences inmycolic acid structure may affect the fluidity and  permeability of an asymmetric lipid bilayer, that would explain the different sensitivity levels of variousmycobacterial species to lipophilic inhibitors [11].It was showed by LIU et al.  [12] that mycolic acid structure plays a critical role in controlling the cell wallfluidity as well as permeability of mycobacteria. They used differential scanning calorimetry to demonstrate that the hightemperature phase transition observed in purified cell walls,usually in the 60-70 o C range, suggestive of a lipid enviroment of extremely low fluidity, can also be observed in whole organisms and in cell walls from which much of the free lipids was removed by extraction with Triton X-114.A survey of seven mycobacterial species demonstrated thatthis high temperature transition was a general property of these organisms. The transition temperature was directlycorrelated to the length of mycolic acid. They have further identified the structural motifs in the mycolic acid molecule,which contribute to the impermeability and the intrinsicdrug-resistance of mycobacteria. Enzyme(s) that catalyze theintroduction of these structural motifs will provide targetsfor the design of new agents for chemotherapy of mycobacterial infections.The mycobacterial mycolic acids are distinguishable fromthose of other genera (such as Corynebacterium ,  Nocardia ,  Rhodococcus ) because they are the largest (C 70  to C 90 ); theyhave the largest α -branch (C 20  to C 25 ); they contain one or two groups (which may be double bonds or cyclopropanerings) that are capable of producing “kinks” in the moleculein the main chain - the meromycolic acid moiety; they maycontain additional oxygen functions to the β -hydroxy group;and, they may have methyl branches in the main carbon backbone [11].According to BARRY III et al.  [13], the modifications inthe mycobacterial mycolic acids occur at two points in themero chain, referred to as distal (closest to the ω -end of thechain) and proximal (closest to the β -hydroxy acid). Thesetwo sites roughly divide the chain into thirds. The twoexceptions to this general pattern are the shorter α ’-mycolates and the ω -1 methoxymycolates of some rapidly-growing species. Polar modifications tend to be restricted tothe distal position and include such functional groups as   An Approach for the Rational Design of New Antituberculosis AgentsCurrent Drug Targets, 2001  , Vol. 2, No. 4   429 methyl ethers, ketones, esters and epoxides. Non-polar modifications occur at both the distal and the proximal positions and include cis  or trans  double bonds and cis  or  trans  cyclopropanes. Trans  functionalities have an adjacentmethyl branch.The most widespread of the classes of mycolic acids arethe α -mycolates, which may contain double bonds or cyclopropanes in cis  configurations or double bonds or cyclopropanes in trans  configurations with an adjacentmethyl branch. Every mycobacterial species produces α -mycolates. The next most widely distributed class of mycolic acids are the ketomycolates, that are found in manydifferent species, regardless of growth rate or status as pathogens or saprophytes. α ’ and wax ester mycolates arealso widely distributed, although they are notably absent in  M. tuberculosis . Epoxymycolates are considerably morerestricted in occurrence. Methoxymycolates appear to be present only in pathogenic species, and primarily in slow-growing. They appear to co-occur in species which also produce ketomycolates. The ω -1 methoxymycolate species isthe most restricted [13]. α -Mycolates, methoxymycolates and ketomycolates arethe three classes of mycolic acids produced by  M.tuberculosis , that differ primarily in the presence and natureof oxygen-containing substituents in the distal portion of themeromycolate branch [13,14] (Fig. 2 ). Small amounts of hydroxymycolic acid were detected in  M. bovis BCG and  M.tuberculosis  [15]. The structure of this molecule, determined  by NMR spectroscopy, mass spectrometry and stereo-chemical studies, strongly suggests that there is a direct biosynthetic relationship between the keto- and the hydroxy-mycolic acids. The knowledge of the biosynthesis of mycolic acids as well as the study of the enzymes involved in this biosynthetic pathway are the keys for the rationaldesign of new antituberculosis agents.A homologous sequence from the  M. leprae  genomesequencing project was used to identify the protein involved in construction of the proximal cyclopropane from  M.tuberculosis [16]. This enzyme (CMAS-2) is the fourthidentified member of a family of proteins to catalyze thetransfer of a methylene group from S  -adenosyl-L-methionineto the double bond of a fatty acid substrate. The threemycobacterial members of this family are closely related toone another, with the cma 2  genes from  M. leprae  and  M.tuberculosis  being more closely related to one another (73%identity) than to the cma 1  gene of  M. tuberculosis  (52%identity). Heterologous expression of cma 2  in  M. smegmatis results in a proportion of the α -mycolates becomingcyclopropanated at the proximal position. Expression of cma1  results in cyclopropanation at the distal position, while co-expression of both genes results in the production of a Fig. (1).  Mycobacterial cell wall.  430   Current Drug Targets  , 2001  , Vol. 2, No. 4Pasqualoto and Ferreira dicyclopropyl mycolate nearly identical to the major mycolicacid produced by  M. tuberculosis . The results of this work suggested that cyclopropanation contributes to the structuralintegrity of the cell wall complex.A common mechanism for the biosynthesis of methoxyand cyclopropyl mycolic acids in  M. tuberculosis  was proposed by YUAN & BARRY III [14] (Fig. 3 ). Themethoxymycolate series has a methoxy group adjacent to amethyl branch, in addition to a cyclopropane in the proximal position. Using the gene for the enzyme that introduces thedistal cyclopropane ( cma 1) as a probe, they cloned and sequenced a cluster of genes coding for four highlyhomologous methyl transferases ( mma  1-4). The mma 4 gene product (MMAS-4) catalyses an unusual S  -adenosyl-L-methionine-dependent transformation of the distal cis -olefininto a secondary alcohol with an adjacent methyl branch.MMAS-3 O -methylates this secondary alcohol to form thecorresponding methyl ether, and MMAS-2 introduces a cis -cyclopropane in the proximal position of the methoxy series.The function of MMAS-1 remained a cryptic issue as it may be functional but lacks the apropriate substrate to catalyzemethyl transfer.Yuan et al.  [17] also reported that MMAS-1 may act at acomplex branch point where expression of this enzymedirectly affects the keto to methoxy ratio in  M. tuberculosis .Overexpression of MMAS-1 has shown to result in theoverproduction of trans -cyclopropane and trans- olefin-containing oxygenated mycolic acids. MMAS-1 converted a cis -olefin into a trans -olefin with concomitant introductionof an allylic methyl branch in a precursor to both themethoxy and ketone-containing mycolic acids (Fig. 4 ). The proportion of mycolic acid containing trans- substituents atthe proximal position of the meromycolate chain is animportant determinant of fluidity of the mycobacterial cellwall and is directly related to the sensitivity of mycobacterialspecies to hydrophobic antibiotics. Inhibiting MMAS-1function in intact cells would be predicted to have the effectof inhibiting trans -mycolate formation and indirectlyketomycolate synthesis, and thereby offers the potential for significant bacteriostatic effects.Mdluli et al.  [18] suggested that an acyl carrier protein(AcpM) is involved in a type II fatty acid synthetase (FAS),which produces the meromycolate branch of full-lengthmycolic acids. AcpM is apparently the carrier for lipids up to50 carbons in length. They also verified that the marked up-regulation of AcpM and β -ketoacyl ACP synthase (KasA,expression of the kasA  gene) accompanying the inhibition of mycolic acid synthesis implies the existence of a regulatorymechanism responsive to the levels of meromycolate or mycolate produced. Such a regulatory system could beexploited to construct a model for the rapid screening of anovel inhibitors of these critical constituents of themycobacterial cell wall. Moreover, these researchers [18]sequenced the kasA  gene in entirety from a geneticallydiverse panel of INH-resistant and INH-susceptible strains,and the results provided compelling evidence that kasA coding sequence alterations participate in the development of INH resistance in the course of human antituberculosis drugtherapy.In the Yuan et al.  [19] study about the biosynthesis of mycolic acids in  M. tuberculosis , they suggested thatmycolate modification reactions occur parallel with the Fig. (2).  Mycolate class distribution in  Mycobacterium tuberculosis .   An Approach for the Rational Design of New Antituberculosis AgentsCurrent Drug Targets, 2001  , Vol. 2, No. 4   431 Fig. (3).  The fate of a common cationic intermediate determines the mycolic acid product formed 14 . Fig. (4). Biosynthetic pathway for cis-  and   trans- oxygenated mycolates and the function of the MMAS-1 gene product 17 .
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