Attacking ATP synthase- Strategy against TB

Greetings

    Looks like am back in the blog world. I was just browsing through my past posts in the blog. Roughly much of the posts has been on viruses specially HIV and Influenza, followed by some Bacteriology. But there is one Bacteriology topic that probably should have been given more importance in this platform. Want to make a guess? Am talking about the world renowned infection- Tuberculosis, simply known as TB. This probably is the right time to bring up this topic, since I have a couple of papers to discuss regarding this topic.

Photo 1: A patient with advanced pulmonary TB

in a tuberculosis hospital in Mumbai. Source

    As always, I had prefer to start of with some basic background introduction so as to bring the point as to what is important and then move on to the most important findings that is of relevance. The infection is caused by Mycobacterium tuberculosis a member of Mycobacterium group. Unlike other bacterial infections, it is an exceptionally difficult to treat organism. Moreover traditional methods of diagnosis such as culture and biochemical analysis takes roughly 3-4 weeks, even in the best of the conditions. All thanks to the slow growing nature of the organism. An estimated 8.7 million people fell ill with TB and 1.4 million died from TB in 2011. The Same source also reports that estimated 20 million lives are saved through use of DOTS and the Stop TB Strategy recommended by WHO.(Reference: WHO).

    There exists a very general idea of how an antibiotic should work (Considering all the class of Antimicrobial drugs). Antibiotic is most effective, if the organism is biochemically active. That means to say, it is very difficult to target drugs against a sluggish or completely inert lying organism. This exactly is the problem in treating several different infections like HIV (Dormant genome, proviral genome), Malaria (Hypnozoites of plasmodium) and of course TB (Absolutely sluggish biochemistry and replication). For starters that is a problem big enough to deal with. The primary line of recommended anti-TB drugs are Isoniazid, Rifampicin, Pyrazinamide, Ethambutol, and Streptomycin. See Table 1.

Table 1: First line drugs used in TB

       First let me consider a best case scenario. A person suffering from cough for more than 2 weeks, is screened for TB by traditional methods such as Acid fast staining of sputum sample, or fluorescence method (such as Auramine O method). More advanced methods such as MGIT culture, luciferase assay, gene detection assays provide better diagnostic support. Newer immune based assays such as IGRA (Interferon Gamma release assay) have slowly replaced mantoux testing. Whatever be the case, once a patient is diagnosed of TB, the subject is involved in a WHO directed program referred as DOTS (Directly Observed Treatment, Short Course). More recently India has adopted DOTS+ strategy for combating MDR and XDR TB. With the best expected patient compliance, it takes almost 6 months to achieve full recovery. Worst case scenario, especially in countries like India, the patient compliance has been not to the mark. When the patient receives treatment, there is a reduction of bacterial load and reduction in symptoms. The patient often discontinues the drug (Assuming recovery, against medical advice), leading to emergence of drug resistant strains. The original treatment strategy involved treating for more than year. With introduction of Pyrazinamide, the treatment time reduced to 6 months (Pyrazinamide can attack semi dormant bacilli also), hence referred as short course treatment.

     

Table 2: Association of TB genes with antibiotic resistance

     There are 2 important problems in the area of TB treatment- MDR and XDR. Multidrug-resistant tuberculosis (MDR-TB) is defined as TB that is resistant to both Isoniazid and Rifampicin, two of the first-line drugs. Extensively drug-resistant TB (XDR TB) is resistant to Isoniazid and Rifampin, plus any fluoroquinolone and at least one of three injectable second-line drugs. The second line injectable drugs include kanamycin, amikacin, capreomycin. Then there is a more recently announced TDR TB, which is misnomer. As per WHO, TDR TB is improperly defined and still susceptible to few drugs (So resistance is not total, though its a superbug).

Fig 1: Structure of Pyridomycin

     So let us put it this way. Our ability to treat TB will depend on 4 factors- Short treatment duration, better patient compliance, New agents with novel mechanisms of action to ensure activity against MDR and XDR and a drug that can attack latent TB infection (LTBI).

   So what have we got in our armory? There is an alternative for INH- "Pyridomycin". It is produced by Dactylosporangium fulvum, and attacks NADH-dependent enoyl- (Acyl-Carrier-Protein) reductase InhA thereby inhibiting mycolic acid synthesis in M. tuberculosis. A huge advantage of this drug is that the clinical isolates that are resistant to INH are susceptible to pyridomycin. That provides one good alternative.

Fig 2: Action of TMC207 on c subunit of
ATP synthase. Source

     But when I think of a novel compound against TB, am reminded of a compound called -TMC207 which was one of the first in pipeline. This is a diarylquinoline compound with a novel mechanism of action- "Inhibition of bacterial ATP synthase". In a study published in NEJM, showed great promise in TB treatment strategy. The mechanism of action is by inhibiting the bacterial ATP synthase, a critical enzyme in the synthesis of ATP for M. tuberculosis. Binding of TMC207 to the oligomeric and proteolipic subunit c of mycobacterial ATP synthase leads to inhibition of ATP synthesis. The gene coding for the c subunit (Link) is atpE with a very highly conserved sequence. A extremely conserved sequence represents difficulty to evolve and thus provides us with a beating advantage. A representation of TMC207 action is shown in Fig 2.

Fig 3: Structure of Q203
Source

    One of the best things that the pharmaceutical companies do very well is to run a very large assay with all the chemicals they have in their chemical library. In doing so, they sometimes come up with a compound that have potential activity against a pathogen which could be of great help. That's what Pethe and his colleagues did. They screened more than 100,000 different chemical compound, of which 106 molecules killed the infectious agent without harming the cells. The best among them- imidazopyridine amide (IPA) was further tested and chemically modified to bring in a brand new chemical Q203.

    The compound was tested in a mouse model. The compound showed good efficacy, better than INH and was well tolerated at a higher dose. My immediate question is what is the mechanism of action. This drug also effects ATP synthesis, but at a different target- cytochrome bc1 complex. From the data that I could gather, Cytochrome bc1 complex looks pretty much conserved sequence.

   A little more of search told me that, this isn't a novel idea. There seems to be a race between many companies. In 2012, there are 2 publications that I could find, which did the same. Abrahams etal, reported Imidazo[1,2-a]pyridine (IP) compound as potential ATP synthesis inhibitor. In another study, Puiying etal identified some chemicals in imidazopyridines (thirteen compounds), Thiophene (one compound) and Benzimidazoles (Nine compounds) as potential ATP synthesis inhibitor. Both studies have zeroed in the compounds by screening a very large set of chemical library using high throughput assays. Claims are that the Q203 is superior to others. The point however is all of them have found Imidazopyridine based compounds as the molecule of interest.

    Probably time for someone to study the efficacy all the drugs together in one parallel study. Definitely, TB is a sluggish organism, but no organism can be sluggish when it comes to ATP synthesis and hence we have an upper hand in this case. The future of Anti- TB looks good.

Hartkoorn RC, Sala C, Neres J, Pojer F, Magnet S, Mukherjee R, Uplekar S, Boy-Röttger S, Altmann KH, & Cole ST (2012). Towards a new tuberculosis drug: pyridomycin - nature's isoniazid. EMBO molecular medicine, 4 (10), 1032-42 PMID: 22987724

Alberto Matteelli, Anna CC Carvalho, Kelly E Dooley, & and Afranio Kritski (2010). TMC207: the first compound of a new class of potent antituberculosis drugs Future Microbiol, 5 (6), 849-858 : PMCID: PMC2921705

Diacon, Andreas H. (2009-06-04) The Diarylquinoline TMC207 for Multidrug-Resistant Tuberculosis., 360 (23), 2397-2405. DOI: 10.1056/NEJMoa0808427

Segala E, Sougakoff W, Nevejans-Chauffour A, Jarlier V, & Petrella S (2012). New mutations in the mycobacterial ATP synthase: new insights into the binding of the diarylquinoline TMC207 to the ATP synthase C-ring structure. Antimicrobial agents and chemotherapy, 56 (5), 2326-34 PMID: 22354303

Pethe etal. Discovery of Q203, a potent clinical candidate for the treatment of tuberculosis. Nature medicine PMID: 23913123

Abrahams KA, Cox JA, Spivey VL, Loman NJ, Pallen MJ, Constantinidou C, Fernández R, Alemparte C, Remuiñán MJ, Barros D, Ballell L, & Besra GS (2012). Identification of novel imidazo[1,2-a]pyridine inhibitors targeting M. tuberculosis QcrB. PloS one, 7 (12) PMID: 23300833

Mak PA, Rao SP, Ping Tan M, Lin X, Chyba J, Tay J, Ng SH, Tan BH, Cherian J, Duraiswamy J, Bifani P, Lim V, Lee BH, Ling Ma N, Beer D, Thayalan P, Kuhen K, Chatterjee A, Supek F, Glynne R, Zheng J, Boshoff HI, Barry CE 3rd, Dick T, Pethe K, & Camacho LR (2012). A high-throughput screen to identify inhibitors of ATP homeostasis in non-replicating Mycobacterium tuberculosis. ACS chemical biology, 7 (7), 1190-7 PMID: 22500615