Structure Activity Relationship Studies for Inhibitors of Type 1 and Type 8 Adenylyl Cyclase Isoforms
Authors: Velasquez Monica Soto , Rana Neha , Alongkronrusmee Doungkamol , Ejendal Karin F.K. , Kyei‐Baffour Kwaku , Van Rijn Richard M. , Dai Mingji , Lill Markus A. , Watts Val J. 
Published: November 2023

Calcium/calmodulin‐stimulated adenylyl cyclase (AC) isoforms AC1 and AC8 are potential targets for treating chronic pain, opioid dependence, and anxiety. The objective of our study was to carry out a series of structure activity relationship (SAR) studies on selective inhibitors of AC1 and/or AC8 activity. Our lab has recently identified a selective inhibitor of AC1 activity, W001, that displayed anti‐allodynic properties in a mouse model of inflammatory pain. Moreover, we revealed that the effect of this compound was prevented by co‐administration of forskolin consistent with an in vivo effect on cAMP signaling. Despite these promising results, W001 also potentiated the activity of AC2 suggesting the potential for unwanted side effects. In an effort to achieve greater overall AC selectivity we undertook two approaches. In the first approach, we performed SAR studies using both commercially available and novel synthesized analogs of W001. The ability of these compounds to inhibit A23187 (calcium ionophore)‐stimulated AC1 activity was examined in HEK cells stably expressing AC1. Active compounds were subsequently evaluated on the closely related AC8 isoform and other representative ACs (i.e. AC2 and AC5). The SAR studies for W001's analogs revealed important structural elements that are important for its inhibitory activity as well as for its selectivity for AC1. Several W001 analogs showed inhibitory activity on both AC1 and AC8, and to date we identified one analog with modest selectivity for AC8 over AC1. This new tool compound may allow us to probe for the role of AC8 in mouse behavioral models. In the second approach toward identifying AC1 inhibitors, we completed a 10,000‐compound screen. We identified two promising scaffolds that have robust AC1 inhibitory activity with IC50 values in the low micromolar range (2–10 μM). These novel and diverse scaffolds revealed both AC1 selective and dual AC1/AC8 inhibition. Additionally, we have identified structural features that diminish the activity at other AC isoforms (e.g. potentiation of AC2). In addition to the SAR studies, an AC1 homology model was developed to predict the binding mode of W001, its analogs, and our newly identified scaffolds. The comparison of computational homology models of AC1 and AC8, in combination with the AC1 and AC8 SAR analyses, provided information about the inhibitor‐residue interactions responsible for the AC1 selectivity of W001. Lastly, we are evaluating our lead compounds in cellular models with the ultimate goal of testing them in animal models of neuropathic pain and opioid dependence. In conclusion, the SARs and molecular docking results indicate that AC1 and AC8 selective inhibition has a structural relationship; this information can be applied to the design and synthesis of more potent and selective AC inhibitors with drug‐like properties.Support or Funding InformationPurdue EVPRP, Indiana Clinical and Translational Sciences Institute (CTSI) and R21 MH101673/NIMH