Monday, December 9, 2024
12/9/2024
Contact PD/PI: Ablordeppey, Seth Funded Project Summary/Abstract: Numerous studies have indicated that 5-HT7R plays a significant role in various psychophysiological functions such as mood stability, cognitive and motor functions, pain tolerance, sleep patterns, appetite, and thermoregulation. Pre-clinical findings have established the role of 5-HT7R in autism spectrum disorders, Fragile X syndrome (FXS), epilepsy, sleep disorders, neuropathic pain and migraine. Meanwhile, several 5-HT7R agonists, have been reported and their potential use in various CNS conditions are being investigated. However, the beneficial effects of activation or blockade of the 5-HT7R is not often clearly established, primarily due to the lack of selective 5-HT7R agents. Even more critical is the absence of biased ligands that could clarify several controversial observations that relate to the 5-HT7R. Thus, our goal to design, synthesize and pharmacologically evaluate new agents with biased signaling towards G-Protein or β- arrestin signaling pathways hold great promise in understanding the 5-HT7R and its application to the treatment of various CNS disorders. Three specific aims (SAs) are proposed. SA 1 will focus on extending our studies on the lead agents for their drug-like properties including brain penetration, pharmacokinetic, metabolic, and bioavailability assessments and cardiotoxicity predictions (HERG, 5- HT2BR). Based on the metabolic evaluations of lead compound 55933, the synthesis and screening of new compounds is proposed in SA 2. This specific aim will also focus on optimization and design of new agents to address the metabolic stability issues such as aromatization and glucuronidation observed in the preliminary studies. For this reason, aromatization susceptible tetrahydroisoquinoline moiety will be replaced with isoindoline which could not undergo aromatization and the CH2OH group will be replaced with substituents such as -F, -CONH2 to restrict glucuronidation. In addition, exploring the electron donating/ withdrawing (σ values) and hydrophilic/hydrophobic (pi values) space around the THI/isoindoline ring systems with bioisosteric substituents, will reveal any improvements in their drug-like characteristics. Simultaneously, docking studies will be carried out using homology models to identify interactions with the key amino acid residues involved in inducing conformations associated with β-arrestin recruitment to the 5-HT7R. SA 3 will cover functional selectivity studies of lead 5-HT7R ligands for their agonist/antagonist properties and G-Protein or β- arrestin signaling bias followed by evaluation of their effect on sleep architecture and NREM/REM sleep pattern under in vivo conditions. Finally, selected test compounds will be compared with SB269970 (5-HT7R antagonist) and compound 1g, a 5-HT7R partial agonist (as a positive control) for their effect on NREM/REM sleep pattern in a mouse model. Progress Report Summary of Parent Award: The full progress report will be submitted and only a brief summary is provided here. In year 2, our primary focus has remained on aims 1 and 2 and involved obtaining selective and functionally biased analogs for evaluation at 5-HTRs. We have designed, synthesized, separated enantiomers, determined the absolute configuration by x-ray crystallography and evaluated another six analogs of lead compounds for binding affinity and functional activity at 5-HT and other CNS receptors. In addition, we have synthesized and evaluated over twenty analogs which have been characterized. A major finding is reported in a manuscript titled: Bricker, B. A.; Voshavar, C.; Onyameh, E. K.; Gonela, U. M.; Lin, X.; Swanson, T. L.; Kozell, L. B.; Schmachtenberg, J. L.; Bloom, S. H.; Janowsky, A. J.; Ablordeppey, S. Y., Enantiomeric Separation, Absolute Configuration by X-ray Crystallographic Analysis, and Functional Evaluation of Enantiomers of the Dual Ligand, SYA0340 at 5-HT(1A) and 5-HT(7A) Receptors. ACS Omega 2023, 8 (24), 21736-21744. In addition, we have applied to the USPTO to protect our intellectual property for several of the compounds under the title: Design and discovery of a high affinity, selective and β-arrestin biased 5-HT7 Receptor Agonist. Project Summary/Abstract Page 6 Contact PD/PI: Ablordeppey, Seth Scientific Justification of Supplement Request A Waters Alliance HPLC/PDA/fraction collector system is needed to supplement an 18.5 year old analytical HPLC system that has lost almost all of it's capability, including having only one pumping chamber left which reduces capability down to only isocratic methods, only manual collection is left, and it has numerous maintenance problems occurring at this point in the instrument's life, causing a work stoppage, and slowing down severely the progress of our students and research staff. The new system will enhance the throughput of the identification, separation, and collection of enantiomers from our chiral drug discovery novel compounds using analytical size HPLC columns. The isolated enantiomers and other test compounds from this system will be used in studies of test compounds and metabolites in the plasma, and brains of rodents from brain penetration and pharmacokinetic studies, metabolic profiling, and bioavailability assessments in support of Specific Aim 1. It will also be used to assess the purity and confirm the synthesis of new compounds in support of Specific Aim 2. It is needed to complete specific aims (SA1-2) as previously stated in the funded grant. This will provide an enhanced capability for our lab and ready access for our diverse graduate students and researchers to use for their PhD projects and research in support of (SA1-2). The entire purchase price of the selected HPLC/PDA/fraction collector system is $99,488.21. We find this price to be reasonable, as shown by the 3 quotes below. Other quotes are $105,429.43 and $123,571.35. All quotes contain academic discounts. This purchase is incurred specifically for the current grant and therefore the cost is 100% allocable to this grant. This equipment is necessary and central to the success of the project to assess the purity and confirm compound identification from the synthesis of new test compounds (SA2). The equipment is also necessary to produce purified test compounds for our pharmacokinetic and brain penetration studies for this project (SA1). The current amount of unobligated grant funds is estimated to be zero dollars ($0) by the end of the second cycle March 31st, 2024. The expenditure plans are to continue to partially support a research associate on the grant and to purchase needed chemicals, support the binding and functional studies of compounds obtained, and evaluate them in animals. There is thus, a clear and dire need for additional funds to acquire this needed equipment to enable the delivery of the specific aims of this research. Future Maintenance of Equipment For the first 3 years, the instrument is covered by a warranty or maintenance agreement and includes a performance maintenance (PM) visit. Training will be provided, and the user will be able to fix most problems after that. Subsequent grant submissions in out years will budget for maintenance of the equipment. Training will be provided during installation. HPLC grade solvents and chemicals will be purchased as needed using existing funds. ****************************************************************************************** Project Summary/Abstract Page 7
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