Elucidating the function of hypothetical PE_PGRS45 protein of Mycobacterium tuberculosis as an oxido-reductase: a potential target for drug repurposing for the treatment of tuberculosis

Abstract

Mycobacterium tuberculosis (Mtb) encodes a total of 67 PE_PGRS proteins and definite functions of many of them are still unknown. This study reports PE_PGRS45 (Rv2615c) protein from Mtb as NADPH dependent oxido-reductase having substrate specificity for fatty acyl Coenzyme A. Computational studies predicted PE_PGRS45 to be an integral membrane protein of Mtb. Expression of PE_PGRS45 in non-pathogenic Mycobacterium smegmatis, which does not possess PE_PGRS genes, confirmed its membrane localization. This protein was observed to have NADPH binding motif. Experimental validation confirmed its NADPH dependent oxido-reductase activity (Km value = 34.85 ± 9.478 μM, Vmax = 96.77 ± 7.184 nmol/min/mg of protein). Therefore, its potential to be targeted by first line anti-tubercular drug Isoniazid (INH) was investigated. INH was predicted to bind within the active site of PE_PGRS45 protein and experiments validated its inhibitory effect on the oxido-reductase activity of PE_PGRS45 with IC₅₀/Ki values of 5.66 μM. Mtb is resistant to first line drugs including INH. Therefore, to address the problem of drug resistant TB, docking and Molecular Dynamics (MD) simulation studies between PE_PGRS45 and three drugs (Entacapone, Tolcapone and Verapamil) which are being used in Parkinson’s and hypertension treatment were performed. PE_PGRS45 bound the three drugs with similar or better affinity in comparison to INH. Additionally, INH and these drugs bound within the same active site of PE_PGRS45. This study discovered Mtb’s PE_PGRS45 protein to have an oxido-reductase activity and could be targeted by drugs that can be repurposed for TB treatment. Furthermore, in-vitro and in-vivo validation will aid in drug-resistant TB treatment.

HIGHLIGHTS

• In-silico and in-vitro studies of hypothetical protein PE_PGRS45 (Rv2615c) of Mycobacterium tuberculosis (Mtb) reveals it to be an integral membrane protein

• PE_PGRS45 protein has substrate specificity for fatty acyl Coenzyme A (fatty acyl CoA) and possess NADPH dependent oxido-reductase activity

• Docking and simulation studies revealed that first line anti-tubercular drug Isoniazid (INH) and other drugs with anti-TB property have strong affinity for PE_PGRS45 protein

• Oxido-reductase activity of PE_PGRS45 protein is inhibited by INH

• PE_PGRS45 protein could be targeted by drugs that can be repurposed for TB treatment

Communicated by Ramaswamy H. Sarma

Keywords:

• PE_PGRS45 protein
• NADPH dependent oxido-reductase
• fatty acyl coenzyme A dehydrogenase
• Mtb drug target
• repurposed TB drug target

Acknowledgement

Medha is a recipient of Senior Research Fellowship from Council of Scientific and Industrial Research (CSIR), Government of India. We greatfully acknowledge Professor Vikas Jain of Indian Institute of Science Education and Research (IISER), Bhopal for his guidance and help in conducting MD simulation studies. We greatfully acknowledge Dr. Neeraj K. Saini, Ramanujan Fellow in conducting the localization studies with recombinant M. smegmatis Jawaharlal Nehru University, New Delhi, India.

Disclosure statement

The authors have no competing interests to declare.

Author’s contributions

Monika Sharma and Medha contributed equally in conceptualization, methodology and writing of the manuscript. Medha conducted all the experiments. Hemant Joshi conducted the localization studies with recombinant M. smegmatis. Monika Sharma and Sadhna Sharma contributed in reviewing and editing of the manuscript.

Additional information

Funding

The financial support by Department of Science and Technology (DST), Government of India (Grant Sanction no. EMR/2016/006774) awarded to Dr. Monika Sharma is gratefully acknowledged.