Pharmacological and genetic activation of cAMP synthesis disrupts cholesterol utilization in Mycobacterium tuberculosis

Abstract

There is a growing appreciation for the idea that bacterial utilization of host-derived lipids, including cholesterol, supports Mycobacterium tuberculosis (Mtb) pathogenesis. This has generated interest in identifying novel antibiotics that can disrupt cholesterol utilization by Mtb in vivo. Here we identify a novel small molecule agonist (V-59) of the Mtb adenylyl cyclase Rv1625c, which stimulates 3’, 5’-cyclic adenosine monophosphate (cAMP) synthesis and inhibits cholesterol utilization by Mtb. Similarly, using a complementary genetic approach that induces bacterial cAMP synthesis independent of Rv1625c, we demonstrate that inducing cAMP synthesis is sufficient to inhibit cholesterol utilization in Mtb. Although the physiological roles of individual adenylyl cyclase enzymes in Mtb are largely unknown, here we demonstrate that the transmembrane region of Rv1625c is required during cholesterol metabolism. Finally, the pharmacokinetic properties of Rv1625c agonists have been optimized, producing an orally-available Rv1625c agonist that impairs Mtb pathogenesis in infected mice. Collectively, this work demonstrates a role for Rv1625c and cAMP signaling in controlling cholesterol metabolism in Mtb and establishes that cAMP signaling can be pharmacologically manipulated for the development of new antibiotic strategies.

Iron limitation in M. tuberculosis has broad impact on central carbon metabolism

“…3 ). We included the frontline drugs isoniazid (INH) and ethambutol (EMB), as well as another compound, mCLB073, an analog of previously described cholesterol inhibitors 27 , 29 , as comparators. PCA indicated that sAEL057 induced the largest change relative to DMSO controls (Fig.…”

“…3 ). mCLB073, belongs to a group of compounds known to activate adenylate cyclase in Mtb encoded by the gene rv1625c , and this leads to inhibition of cholesterol catabolism 27 , 29 . Intriguingly, PCA analysis showed a clear separation between sAEL057- and mCLB073-treated Mtb despite both compounds showing the highest activity in the presence of cholesterol, suggesting that the compounds differ in their MOA (Fig.…”

Cyclic AMP is a critical mediator of intrinsic drug resistance and fatty acid metabolism in M. tuberculosis

“…Consistent with the role of cAMP in modulating drug sensitivity, overexpression of rv1339 but not the catalytically dead rv1339 allele resulted in increased vancomycin sensitivity in Mtb ( Figure 5F ). To demonstrate that modulating cAMP levels is sufficient to regulate Mtb H37Rv antibiotic sensitivity, we treated rv3645 knockdown strains with V-59, a small molecule agonist of the adenylate cyclase Rv1625c that results in constitutive cAMP production (Wilburn et al, 2022). Addition of V-59 rescued the vancomycin sensitivity of rv3645 knockdown strains ( Figure 5G ).…”

“…cAMP levels are coordinately regulated by Rv3645 and the atypical phosphodiesterase Rv1339. cAMP produced by Rv3645 is critical for Mtb growth on long-chain fatty acids, a host-relevant carbon source, and for intrinsic multidrug resistance, highlighting the potential utility of small molecule modulators of this second messenger to control Mtb infection (Wilburn et al, 2022).…”

“…The adenylate cyclase Rv1625c has recently been shown to be important for cholesterol catabolism, although interestingly this phenotype was independent of the Rv1625c cyclase homology domain (Wilburn et al, 2022). In a screen for compounds that disrupt cholesterol catabolism, VanderVen and colleagues discovered V-59, an Rv1625c agonist analogous to the eukaryotic adenylate cyclase activator forskolin (VanderVen et al, 2015; Wilburn et al, 2022). V-59 results in constitutive activation of Rv1625c and cAMP production and, for reasons yet unknown, blocks cholesterol catabolism.…”