Millions of deaths caused by Mycobacterium tuberculosis (Mtb) are reported worldwide every year. Treatment of tuberculosis (TB) involves the use of multiple antibiotics over a prolonged period. However, the emergence of resistance leading to multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB) is the most challenging aspect of TB treatment. Therefore, there is a constant need to search for novel therapeutic strategies that could tackle the growing problem of drug resistance. One such strategy could be perturbing the functions of novel targets in Mtb, such as universal stress protein (USP, Rv1636), which binds to cAMP with a higher affinity than ATP. Orthologs of these proteins are conserved in all mycobacteria and act as “sink” for cAMP, facilitating the availability of this second messenger for signaling when required. Here, we have used the cAMP-bound crystal structure of USP from Mycobacterium smegmatis, a closely related homolog of Mtb, to conduct a structure-guided hunt for potential binders of Rv1636, primarily employing molecular docking approach. A library of 1.9 million compounds was subjected to virtual screening to obtain an initial set of ~2,000 hits. An integrative strategy that uses the available experimental data and consensus indications from other computational analyses has been employed to prioritize 22 potential binders of Rv1636 for experimental validations. Binding affinities of a few compounds among the 22 prioritized compounds were tested through microscale thermophoresis assays, and two compounds of natural origin showed promising binding affinities with Rv1636. We believe that this study provides an important initial guidance to medicinal chemists and biochemists to synthesize and test an enriched set of compounds that have the potential to inhibit Mtb USP (Rv1636), thereby aiding the development of novel antitubercular lead candidates.

Mycobacterium tuberculosis employs several signaling pathways to regulate its cellular physiology and survival within the host. Mycobacterial genomes encode multiple adenylyl cyclases and cAMP effector proteins, underscoring the diverse ways in which these bacteria utilize cAMP. We have earlier identified universal stress proteins (USP), Rv1636 and MSMEG_3811 in M. tuberculosis and M. smegmatis respectively, as abundantly expressed, novel cAMP-binding proteins. In this study, we show that these USPs may function to regulate cAMP signaling by direct sequestration of the second messenger. In slow-growing mycobacteria, concentrations of Rv1636 were equivalent to the amounts of cAMP present in the cell, and overexpression of Rv1636 in M. smegmatis increased levels of 'bound' cAMP. Rv1636 is secreted via the SecA2 secretion system in M. tuberculosis but is not directly responsible for the efflux of cAMP from the cell. While msmeg_3811 could be readily deleted from the genome of M. smegmatis, we find that the rv1636 gene is essential for growth of M. tuberculosis, and this functionality depends on the cAMP-binding ability of Rv1636. This is the first evidence of a 'sink' for any second messenger in bacterial signaling that would allow mycobacterial cells to regulate the available intracellular 'free' pool of cAMP.