Protein kinase C-δ inhibitor, Rottlerin inhibits growth and survival of mycobacteria exclusively through Shikimate kinase

Highlights

• Shikimate Kinase (SK) is an essential protein for survival of mycobacteria.

• SK of pathogenic and non-pathogenic mycobacteria are structurally different.

• Rottlerin inhibits growth and survival of pathogenic mycobacteria through SK.

• Rottlerin does not affect non-pathogenic mycobacteria due to weak binding with SK.

Abstract

The molecular bases of disease provide exceptional prospect to translate research findings into new drugs. Nevertheless, to develop new and novel chemical entities takes huge amount of time and efforts, mainly due to the stringent processes. Therefore, drug repurposing is one of such strategies which is being used in recent times to identify new pharmacophores. The essential first step in discovery of the specific inhibitor with low toxicity is the identification and elucidation of pathways exclusive to target pathogen. One such target is the shikimate pathway, which is essential for algae, higher plants, bacteria and fungi. Since, this enzyme system is absent in higher eukaryotes and in mammals, the enzymes involved in the pathway provide an attractive target for the development of potentially selective and non toxic antimicrobial agents. Since, so far there is no specific inhibitor which is able to restrain mycobacterial shikimate pathway; we expanded the use of a known kinase inhibitor; Rottlerin, in order to predict the prototype in discovering the specific molecules against this enzyme. For the first time we have shown that Rottlerin inhibits extracellular mycobacteria by affecting Shikimate Kinase (SK) and this effect is further enhanced during the intracellular infection due to the added effect of PKC- δ down-regulation. The molecular docking of Rottlerin with both the mycobacterial SKs, corroborated the inhibition data, and revealed that the effects of SK, in slow and in fast grower mycobacteria are due to the changes in affinity of binding with the drug.

Introduction

Tuberculosis (TB) is a respiratory disease and a leading cause of death worldwide. Despite the availability of effective chemotherapy and protective vaccine, TB is still a major concern and threat to global health, which demands the urgent need to identify new drugs and their targets that can shorten the regimen period and pill burden [1] and overcome with the problem of drug resistance. This should provide insight in development of newer compounds and repurposing of the existing standard drugs that may help in combating this problem. Repurposing of the existing drugs is a better option, as it saves the ample amount of money, time and effort due to the stringent processes required in the drug development program. The translation of the existing approved drugs is quicker and less expensive in comparison to newer drug discovery, as the cytotoxicity, pharmacology and formulation of such compounds are already known.

Since, MTB has around 700 essential proteins, which are either involved in pathogenesis or in multiplication of the bacteria, it seems imperative to search for alternate mechanism(s) to target in order to find out new molecules. One such promising target is shikimate pathway which is essential in algae, higher plants, bacteria, and fungi, but is absent in mammals [2], [3]. The bacterial shikimate pathway is crucial for the synthesis of many important metabolites including aromatic amino acids, folic acid, and quinones, whereas mammals must obtain most aromatic compounds from their diet [3]. For example, MTB growth in macrophages has been shown to require mycobactin siderophores derived from the shikimate pathway. One of the enzymes of this pathway, Shikimate kinase (SK) is crucial for mycobacterial growth [4] and is the fifth enzyme in the pathway that catalyzes phosphate transfer from ATP to the 3-hydroxyl group of shikimic acid, producing shikimate-3-phosphate [2]. Mycobacterium SK (MTSK) is encoded by arok gene and the ortholog of this enzyme is also present in non- pathogenic strain i.e in Mycobacterium smegmatis (MSSK). SK belongs to the family of nucleoside monophosphate (NMP) kinase and consists of LID, CORE and NMP-binding domains [[5], [6]. Besides this, the enzyme is a validated drug target for anti-tubercular drug, and a large pool of active inhibitors has been designed and screened by targeting this enzyme. However, none of the molecules has gone for discovery pipeline, till date. Considering this, and owing to the absence of shikimate kinase pathway in mammalian system, SK is thus considered to be an essential target for the development of the specific inhibitors with least expected cross reactivity with the host systems.

In the recent years polyphenol compounds are being thoroughly considered in drug discovery program. Action of polyphenols in cancer biology has been well established. It can induce tumor cell death and can interfere with the dissemination, carcinogenesis and tumor growth [7]. Recently in few of the studies, polyphenols from different sources have been screened against mycobacteria and have shown promising results [8], [9]. These compounds are safer and metabolize better than the pharmaceutical drugs, as these compounds are derived from the natural sources [10]. Therefore, subsequent to our understandings regarding the importance of polyphenols and SK; in the present manuscript we have proposed one of the polyphenols; Rottlerin, to look for its effect against mycobacteria with special reference to its SK. This compound is also an inhibitor of Protein kinase C-δ. Molecular interactions and homology modeling of Rottlerin with SK have shown that the enzyme from slow grower binds better to the inhibitor compared to the SK from fast grower non-pathogenic mycobacteria.