Proteome-wide Profiling of Isoniazid Targets in <em>Mycobacterium tuberculosis</em>

Argyrides Argyrou

Lianji Jin

Linda Siconilfi-Baez

Ruth Angeletti

John Blanchard

^{Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.}

^{Laboratory for Macromolecular Analysis & Proteomics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.}

^{Present address: Department of Chemical Enzymology, Bristol-Myers Squibb Pharmaceutical Company, Pharmaceutical Research Institute, P.O. Box 5400, Princeton, NJ 08543-5400, USA.}

^{To whom correspondence should be addressed: John S. Blanchard Department of Biochemistry Albert Einstein College of Medicine 1300 Morris Park Avenue Bronx, NY 10461 USA Tel: (718) 430-3096; Fax: (718) 430-8565; E-mail:}ude.uy.mocea@rahcnalb

Abstract

Isoniazid (INH) is an essential drug used to treat tuberculosis. The mycobactericidal agents are INH adducts (INH-NAD(P)) of the pyridine nucleotide coenzymes, which are generated in vivo after INH activation, and which bind to, and inhibit, essential enzymes. The NADH-dependent enoyl-ACP reductase (InhA) and the NADPH-dependent dihydrofolate reductase (DfrA) have both been shown to be inhibited by INH-NAD(P) adducts with nanomolar affinity. In this paper, we profiled the M. tuberculosis proteome using both the INH-NAD and INH-NADP adducts coupled to solid supports and identified, in addition to InhA and DfrA, sixteen other proteins that bind these adducts with high affinity. The majority of these are predicted to be pyridine nucleotide-dependent dehydrogenases/reductases. They are involved in many cellular processes including S-adenosylmethionine-dependent methyl transfer reactions, pyrimidine and valine catabolism, the arginine degradative pathway, proton and potassium transport, stress response, lipid metabolism, and riboflavin biosynthesis. The targeting of multiple enzymes could, thus, account for the pleiotropic effects of, and powerful mycobactericidal properties of, INH.

Abstract

The powerful and specific antitubercular effects of isoniazid (isonicotinic acid hydrazide, INH) were discovered in 1952, and revolutionized the treatment of tuberculosis (1-3). INH continues to be extensively used in the treatment of the disease to this day; singly in prophylaxis or in a multi-drug combination with rifampicin, pyrazinamide and ethambutol for active infections.

The mechanism by which this simple compound exerts its powerful effect on Mycobacterium tuberculosis (minimum inhibitory concentration, MIC = 0.02−0.05 μg ml^{) began to be unraveled in the early 1990's shortly after genetic tools for mycobacteria were being developed. Strains of}M. tuberculosis and Mycobacterium smegmatis that were resistant to high levels of INH were first shown to have deletions or point mutations in the katG gene, which led to the proposal that INH was a pro-drug that was oxidatively activated by the katG-encoded mycobacterial catalase-peroxidase (4). In a second genetic study, a spontaneous M. smegmatis mc^{155 mutant and a}Mycobacterium bovis BCG mutant that were co-resistant to INH and ethionamide, a structural analog of INH, were both shown to have a point mutation (S94A) in the inhA-encoded enoyl-ACP reductase (5). Furthermore, resistance was observed through overexpression of wild-type InhA or the S94A mutant in M. smegmatis mc^{155, which led to the identification of InhA as a target for INH (}5).

A consensus on the mechanism of action of, and resistance to, INH has emerged over the past few years. The oxidation of INH by KatG generates an isonicotinoyl radical that reacts non-enzymatically with cellular pyridine nucleotides to generate an ensemble of isonicotinoyl-NAD(P) adducts (INH-NAD(P); Figure 1) (6). Of the twelve possible adducts of INH, only two have thus far been shown to inhibit essential enzymes. The acyclic 4S isomer of INH-NAD (compound 1, Figure 1) is known to be a slow-onset, tight-binding inhibitor of InhA (K_i^{= 1 nM) (}7,8). Inhibition of InhA prevents the elongation of C₂₆ fatty acids by the fatty acid synthase II complex (9,10), and prevents the formation of the mycolic acids that are an important defense against the host immune surveillance and defense system (11,12). We have recently demonstrated that the acyclic 4R isomer of INH-NADP (compound 4, Figure 1) inhibits the dfrA-encoded dihydrofolate reductase (DHFR) with sub-nanomolar affinity, and acts as a bisubstrate analog (13). DHFR maintains the cellular pool of folic acid in the four-electron reduced form by catalyzing the NADPH-dependent reduction of dihydrofolate (and folate, albeit less efficiently) to tetrahydrofolate (14). By acquiring single-carbon units at the methyl, methylene, and formyl oxidation levels, tetrahydrofolate is an important single-carbon coenzyme donor involved in many important enzymatic reactions, which are necessary for the biosynthesis of nucleic acids, purines, pyrimidines, and several amino acids (14).

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FIGURE 1

Chemical structures of the INH-NAD and INH-NADP adducts. The adenosinediphospho-ribose (for INH-NAD) and 2′-phospho-adenosine-diphospho-ribose (for INH-NADP) moiety of these molecules is abbreviated as ADPR(P). Each of the R and S acyclic forms is in equilibrium with the corresponding pair of diastereomeric, cyclic, hemiaminal forms. Cyclization occurs by intramolecular attack of the amide nitrogen of the nicotinamide ring on the carbonyl group of the isonicotinoyl moiety (6).

Clinical resistance to isoniazid has been increasing rapidly leading to treatment failure and diminished therapeutic outcomes. The appearance of multi-drug resistant tuberculosis (resistance to the two first line drugs, isoniazid and rifampicin) requires the lengthy use of second line drugs that are less effective, more toxic, and more costly than the first line drugs (15). Thus, new anti-tubercular drugs are urgently needed. Effective drug development requires good drug target selection. A powerful way to identify such targets is to identify the targets of existing effective drugs, such as INH against tuberculosis.

Are there other INH targets? The majority of INH-resistant M. tuberculosis clinical isolates have mutations in katG or inhA, however, 10–25 % of these have unknown genotypes (15) suggesting that there may be additional targets. In addition, the complete genome sequence of M. tuberculosis (16) suggests that, like other organisms, there are a large number of pyridine nucleotide-dependent reductases and dehydrogenases with substrates that could be mimicked by INH-NAD(P) adducts acting as bisubstrate analogs of these enzymes.

Although mycobacterial genetics has continued to provide insight into the mechanisms of resistance to INH (17-19), it has not yet provided any additional INH targets. Genomic (20-24) and proteomic (25) studies have also been employed to identify genes that show altered levels of expression when mycobacteria are treated with INH. However, the RNA and protein levels of KatG, InhA, and DHFR are unaltered after INH treatment and, to date, these studies have failed to provide any new targets for INH. In this paper we employed the old, but extraordinarily powerful, method of affinity chromatography on solid supports to which the INH-NAD and INH-NADP adducts were coupled to effect the selective purification of those enzymes that bind the INH adducts tightly. In addition to InhA and DHFR, we identified sixteen other proteins that bind these adducts tightly. These newly identified proteins are potential candidates for drug development that require further validation including gene knock-out studies to assess essentiality, overexpression of these proteins in M. tuberculosis to determine if they can confer resistance to isoniazid by drug sequestration, recombinant protein expression and purification, assay development to confirm or assign function and determine the extent of inhibition of enzyme activity by the INH adducts in vitro, and X-ray crystallography structural studies.

ABBREVIATIONS

ACP	acyl carrier protein
DHFR	dihydrofolate reductase
InhA	enoyl-ACP reductase
EDTA	ethylenediaminetetraacetic acid
INH	isonicotinic acid hydrazide (isoniazid)
INH-NAD(P)	isonicotinoylated nicotinamide adenine dinucleotides
NAD(P)⁺	oxidized nicotinamide adenine dinucleotide (phosphate)
NAD(P)H	reduced nicotinamide adenine dinucleotide (phosphate)
Pipes	piperazine-1,4-bis(2-ethanesulfonic acid)
SDS-PAGE	sodium dodecylsulfate polyacrylamide gel electrophoresis
Tris	tris(hydromethyl)aminomethane

ABBREVIATIONS

Footnotes

SUPPORTING INFORMATION AVAILABLE

Proteomic analysis of INH-NAD(P)-binding proteins (Supplementary Table 1). This information is available free of charge via the Internet at http://pubs.acs.org.

Footnotes

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