Abstract
The main and prime objective of this project is to identify new anti-Mycobacterial tuberculosis drugs targeting FadD32 which is a fatty acyl- AMP ligase involved in the biosynthesis of mycolic acids; essential for the survival of Mycobacterium tuberculosis, the causative agent of tuberculosis (Léger et al., 2009). This protein catalyzes the conversion of fatty acid to acyl-adenylate (acyl-AMP) in the presence of adenosine triphosphate and is conserved in all the mycobacterial species sequenced so far, thus representing a promising target for the development of novel anti-tuberculous drugs (Galandrin et al., 2013; Kuhn et al., 2016). The mycolic acid is the key component of cell wall of Mycobacterium tuberculosis and its biosynthesis is a particularly good source of molecular targets because the biosynthetic enzymes do not have homologues in the mammalian system (Jackson et al., 2013).
FadD32 is one of the 34 FadD proteins of M. tuberculosis annotated as putative fatty acyl-CoA synthetases, and its role in mycolic acid biosynthesis is to activate C48–C64 meromycolic acid for condensation by PKS13 (Li et al., 2015). A combination of biochemical and enzymatic approaches demonstrated that FadD32 exhibits substrate specificity for relatively long-chain fatty acids. More importantly, FadD32 catalyzes the transfer of the synthesized acyl-adenylate onto specific thioester acceptors, thus revealing the protein acyl-ACP ligase function. Therefore, FadD32 might be the prototype of a group of M. tuberculosis polyketide-synthase-associated adenylation enzymes possessing such activity. A substrate analog of FadD32 inhibited not only the enzyme activity but also mycolic acid synthesis and mycobacterial growth, opening an avenue for the development of novel antimycobacterial agents (Léger et al., 2009).
Introduction
Drug resistance especially antibiotic resistance is now a universal challenging concern, creating tough hurdle in treating infectious diseases and others, and undermining many advances in health and medicine. Antibiotic resistance occurs naturally and its distribution is also heterogeneous, but misuse of antibiotics in humans and animals is accelerating the process. A review commissioned by UK government emphasis on global action against the antibiotic resistance and warned that by 2050 the superbugs will kill someone every three seconds. The review highlighted this alarming situation as a devastating problem by 2050 responsible for an estimated 10 million deaths a year (Choices, 2016; AMR Review, 2016). Tuberculosis is one of the hotspot lethal disease with high morbidity and mortality rates caused by Mycobacterium tuberculosis affected millions of people and their civilization with a prominent history of epidemics and pandemics. But later and very recently drug resistant Mycobacterium tuberculosis strains emergence in all over the world particularly in developing countries compromising the current treatment strategies (Cloete et al., 2016).
In 2014, 9.6 million people fell ill with TB and 1.5 million died from the disease. Over 95% of TB deaths occur in low- and middle-income countries, and it is among the top 5 causes of death for women aged 15 to 44. In 2014, an estimated 1 million children became ill with TB and 140 000 children died of TB. It is a leading killer of HIV-positive people: in 2015, 1 in 3HIV deaths was due to TB. Globally in 2014, an estimated 480 000 people developed multidrug-resistant TB (MDR-TB). Ending the TB epidemic by 2030 is among the health targets of the newly adopted Sustainable Development Goals (“WHO | Global tuberculosis report 2015”).
Currently there are two categories of drugs available for the treatment of tuberculosis classified into; first line drugs such as, isoniazid (INH), rifampin (RIF), pyrazinamide (PZA), ethambutol (EMB) and second line drugs like para-amino salicylate (PAS), kanamycin, cycloserine (CS), ethionamide (ETA), amikacin, capreomycin, thiacetazone, fluoroquinolones etc. The DOTS (directly observed treatment, short-course) TB therapy comprised of 2 months treatment with 4 drugs, INH, RIF, PZA and EMB daily, followed by 4 months treatment with INH and RIF, three times a week (Lamichhane, 2011).
The most challenging factor in the management, control, and eradication of tuberculosis is the emergence of multi-drug resistance tuberculosis (MDR-TB) and extensive drug resistance tuberculosis (XDR-TB)(Johnson et al., 2006). MDR-TB is resistant to at least isoniazid and rifampicin which are the two most powerful anti-TB drugs while XDR-TB, in addition to being resistance to at least isoniazid and rifampicin, is also resistant to at least three of the six main classes of second-line drugs(“WHO | Global tuberculosis report 2015”). The resistance problem is tackled in the past by employing different strategies including: rotation of antibiotic combinations, identification of less mutable targets, search for new chemical entities for known targets, use of virulence factors as targets and phenotypic conversion, which aims to inhibit the resistance mechanism employed by the bacterium (Tan et al., 2000). Although with using these counter measures, it is stated that resistance still on the rise. Moreover, MDR-TB is treated with second line drugs but due to longer duration, less effective, more toxic, and more expensive than isoniazid and rifampicin creating big hurdles in treatment process.
Therefore a strict emphasis is addressing to investigate new potential and reliable therapeutic targets to overcome the current situation with the discovery of new drugs (Gupta et al., 2001).
To formulate a significant strategy to overcome the current scenario of drug resistant TB problem, it is of prime importance to discover and develop new drugs with novel targets, are to be bactericidal and having low profile of toxicity and must have important aspect of having no homologue in mammalian host. And these drugs should to inhibit the novel targets and must be involved in vital aspects of bacterial growth, metabolism, and viability. These targets could include cell wall synthesis, nucleic acid biosynthesis, protein biosynthesis, and energy metabolism, resulting in either growth inhibition or death of the bacteria (Chopra et al., 2002).
The potential virulence determinant of mycobacterium tuberculosis is cell wall which is composed of three covalently linked macromolecules; peptidoglycan, arabinogalactan and mycolic acids. The cell wall protect M. tuberculosis immune system in infected host. In addition, the genome of M. tuberculosis encodes a series of pathways that are unique in M. tuberculosis but are absent in mammalian cells and also has the capability of inhibition selectively and with minimum side effects. The most important characteristic of cell wall of M. tuberculosis is its criticality for its growth and survival in infected host, and thus, contributes to the resistance to most commonly-used antibiotics and chemotherapeutic agents. The Cell wall is regarded as the best source of molecular targets because the biosynthetic enzymes do not have homologues in mammalian system (Mdluli and Spigelman, 2006). Among these, mycolic acids biosynthesis is the hallmark of Mycobacterium tuberculosis and having potential targets among which the final condensation step of FAS-II pathway is crucial comprising of Polyketide synthase Pks13, Acyl-AMP ligase, FadD32 and AccD4-containing acyl-coenzyme A (CoA) carboxylase (Lou & Zhang, 2010).
Therefore need for new drugs against these drug resistant strains is the key requisite to be designed with an ideal characteristics such as, rapidly bactericidal (rather than bacteriostatic), with novel mechanisms of actions and possess potent sterilizing activity to enable a stable cure to be achieved in a shorter time period than for the currently available therapy (Kaneko et al., 2011; Zuniga et al., 2015).
Problem statement
Drug resistance especially antibiotic resistance is now a challenging problem worldwide which occurs naturally, but misuse of antibiotics in humans, agriculture, and animals is accelerating the process. Therefore, rising resistance rates to current anti-tuberculosis drugs highlight the need for new drugs that have novel mechanisms of action.
Methods
The strategy devised for execution of the project include various steps of computational approaches and wet laboratory experiments. In the first step after thorough literature survey computational studies will be performed for virtual screening of small synthetic compounds against the FadD32 of Mycobacterium tuberculosis. After this, different stepwise experimental procedures will be carried out which included the cloning of FadD32 gene, its expression and purification and then In vitro and In vivo assays. After completion of first round, the catalogue based similarity search analysis of first round most potent hits will be carried out, then the in vitro and in vivo assays of best selected compounds will be employed. Finally co-crystallization by biophysical characterizations will be pushed to achieve the stated goals.
Data Analysis
For data analysis SPSS or some other relevant software will be employed to get the optimized data and thus significant results.
Results Statement
Through these studies, we will be able to uncover the more bioactive chemical space against FadD32 enzyme. These molecules could be pushed further for synthetic optimization that will ultimately lead to the therapeutic interventions against Mycobacterium tuberculosis.
Discussion
This project will provide a solid framework for future understanding of biology in perspective of this target (FadD32) of Mycobacterium tuberculosis. It will result in therapeutic intervention with a new gateway to novel treatment strategies. The project will provide the opportunity for man power development and research activities regarding TB drug discovery and designing (Zuniga et al., 2015).
Although tuberculosis has high morbidity and mortality outcomes worldwide, and is one of the top 10 causes of death. According WHO over 95% of TB deaths occur are occurring in developing countries. So, as the key problem in the developing countries there is urgent need to identify novel antitubercular agents with new mode of action to combat the emerging MDR-TB and XDR-TB strains.
So, overall, the project studies will advance our understanding about this new promising target of mycobacteria and open an avenue for the development of mechanism-based novel antimycobacterial like drugs.
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