Effects of 39 Compounds on Calmodulin-Regulated Adenylyl Cyclases AC1 and Bacillus anthracis Edema Factor

Abstract

Adenylyl cyclases (ACs) catalyze the conversion of ATP into the second messenger cAMP. Membranous AC1 (AC1) is involved in processes of memory and learning and in muscle pain. The AC toxin edema factor (EF) of Bacillus anthracis is involved in the development of anthrax. Both ACs are stimulated by the eukaryotic Ca2+-sensor calmodulin (CaM). The CaM-AC interaction could constitute a potential target to enhance or impair the AC activity of AC1 and EF to intervene in above (patho)physiological mechanisms. Thus, we analyzed the impact of 39 compounds including typical CaM-inhibitors, an anticonvulsant, an anticholinergic, antidepressants, antipsychotics and Ca2+-antagonists on CaM-stimulated catalytic activity of AC1 and EF. Compounds were tested at 10 μM, i.e., a concentration that can be reached therapeutically for certain antidepressants and antipsychotics. Calmidazolium chloride decreased CaM-stimulated AC1 activity moderately by about 30%. In contrast, CaM-stimulated EF activity was abrogated by calmidazolium chloride and additionally decreased by chlorpromazine, felodipine, penfluridol and trifluoperazine by about 20–40%. The activity of both ACs was decreased by calmidazolium chloride in the presence and absence of CaM. Thus, CaM-stimulated AC1 activity is more insensitive to inhibition by small molecules than CaM-stimulated EF activity. Inhibition of AC1 and EF by calmidazolium chloride is largely mediated via a CaM-independent allosteric mechanism.

International Union of Basic and Clinical Pharmacology. CI. Structures and Small Molecule Modulators of Mammalian Adenylyl Cyclases

“…AC1 forms a unique interface with its activator CaM compared with other mammalian CaM targets (Vorherr et al, 1993;Diel et al, 2008;Masada et al, 2012;Lübker and Seifert, 2015;Lübker et al, 2015a,b). Principally, such a unique protein-protein interaction interface could be exploited pharmacologically.…”

“…Principally, such a unique protein-protein interaction interface could be exploited pharmacologically. In this context, it is interesting to note that calmidazolium chloride, which is very potent at inhibiting numerous CaM-regulated effectors, only very ineffectively inhibits AC1 (Lübker and Seifert, 2015). However, potent and TABLE 7 Interaction of prototypical membrane-permeable P-site inhibitors with mACs, sAC, sGC, and adenosine receptors AC activity was determined in the presence of Mg 2+ and in the presence of stimulators (Ga S-GTPgS and/or FSK) to ensure maximum catalytic activity and, hence, highest inhibitor potency possible.…”

“…15) is mechanistically related to the P-site inhibitors shown here. (Lübker and Seifert, 2015). Calmidazolium also inhibits AC9, probably by binding to the catalytic site (Haunso et al, 2003).…”

Alternative pre-approved and novel therapies for the treatment of anthrax

“…Since CaM stimulates EF catalytic activity, certain studies have targeted CaM and the CaM-target interaction [37, 151–153]. Several well-known, potent CaM-inhibitors that have already been discovered and produced for their use in other illnesses, such as depression and psychosis, have demonstrated good efficacy against EF [151, 154–157].…”

A Simple Luminescent Adenylate-Cyclase Functional Assay for Evaluation of Bacillus anthracis Edema Factor Activity

“…As of today, the enzymatic activity of EF is determined by measuring the concentration of the cAMP resulting from the EF-mediated conversion of ATP, either by radioactive labeling with [α- 32 P]-ATP and subsequent chromatographic fractionation for separation of the radioactive cAMP product [11,17,18] or by the use of specific anti-cAMP antibodies [19,20,21]. …”