Authors :
Presenting Author: Rhashanda Haywood, MS – University of Maryland Eastern Shore
Miguel Martin, PhD – Professor, Pharmaceutical Sciences, University of Maryland Eastern Shore; Patrice Jackson-Ayotunde, PhD – Associate Professor, Pharmaceutical Sciences, University of Maryland Eastern Shore
Rationale:
Epilepsy is the fourth common neurological disorder after Parkinson’s and Alzheimer’s Disease and Stroke, with 3 million people in the US and 65 million people worldwide living with epilepsy. Numerous reports have shown that voltage gated Na+ and Ca+2 channels (VGNC, VGCC) are implicated in several molecular mechanisms by which seizures can occur. Blockage of these channels can lower the frequency of seizures. Our lab engages in early drug design and discovery of novel, small molecules as potential anti-seizure analogs for epilepsy. Previous hit-to-lead optimization and target identification studies performed by our research team led to the discovery of lead compound IAA65. This analog has been shown to act as a potent and selective Cav3.2 and Cav3.3 T-type voltage-gated calcium channel (T-VGCC) inhibitor. The objective of the work presented is to evaluate the inhibitory effects of two novel derivatives based on the prototype IAA65 to identify new lead compounds as T-VGCC inhibitors.Methods:
Preliminary in vitro whole cell patch clamp experiments were performed to assess the inhibitory effects of novel analogs RHB56 and RHB59 on voltage-activated Ca2+ currents in human embryonic kidney (HEK-293) cells. Cav3.2 transfected HEK 293 cells were treated with 50 µM of test compounds RHB56 and RHB59 and compared to the control group (none pretreated HEK 293 cells). Statistical analyses consisted of t-test for pairwise comparisons or one-way ANOVA followed by post-hoc analysis using Tukey’s honest significant difference test for comparisons between multiple groups.
Results:
RHB56 at 50 µM caused a significant reduction in the calcium currents generated by the Cav3.2 T-VGCC subunit. No inhibitory effect was shown at a similar concentration for analog RHB59. The effect of RHB56 on calcium currents was concentration dependent, as the compound caused a left-ward shift in the steady-state inactivation of T-VGCC. Analog RHB59 had no effect on the steady state inactivation, the current voltage relationship, or inactivation of T-VGCCs. These results show that RHB56 is a potent inhibitor of T-VGCC by altering channel inactivation. In vitro studies are currently underway on analog, RHB56 to further examine the mechanism of action on T-VGCCs.
Conclusions:
A target-based drug design strategy is currently in progress on lead compound IAA65 to identify new lead compounds that will inhibit T-VGCCs. To validate the biological activity of our novel analogs in preclinical seizure rodent models, studies will be conducted at the National Institutes of Neurological Disorders and Stroke Epilepsy Therapy Screening Program, NIH. Molecule (RHB56) is currently undergoing investigation in acute rodent seizure models, to determine the efficacy and safety as an anti-seizure agent.
Funding:
Pharmaceutical Research and Manufacturers of America Pre-Doctoral Fellowship in Drug Discovery