Browsing by Author "Agogo-Mawuli, Percy"
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Item Computational Pharmacology for Identifying and Refining Novel Inhibitors of the Regulator of G protein Signaling type-12 (RGS12) Protein Target(2023) Agogo-Mawuli, Percy; Siderovski, DavidPurpose: Substance use disorders arise from persistent changes in CNS synaptic transmission, as caused by initial exposure to illicit substances that heighten dopamine levels in the brain’s reward circuitry (a key event in establishing long-term drug-seeking behavior) [1,2]. The Siderovski lab recently discovered that mice lacking Regulator of G protein Signaling-12 (RGS12) are attenuated in their normal hyperlocomotion elicited by acute cocaine, amphetamine, or methamphetamine [3,4]. RGS12-deficient mice have increased dopamine transporter (DAT) expression and increased dopamine uptake within the ventral striatum [3]. The target for RGS12’s action as a Galpha-directed GTPase-accelerating protein (GAP) is the presynaptic kappa opioid receptor (KOR) [4], as KOR activation is known to attenuate striatal dopaminergic tone [5]. Our hypothesis is that RGS12 directly modulates the output of dynorphin / KOR signaling to dopamine reuptake. Developing RGS12 inhibitors would provide complementary pharmacological means to test this hypothesis pre-clinically, including in rodent models. However, to date, there are no small-molecule inhibitors of the G-alpha: RGS domain protein-protein interaction that are not thiol-reactive covalent modifiers of the RGS protein (a highly undesirable chemical property anathematic to further drug development) [6]. Methods: Using the AtomNet® model, a deep convolutional neural network for structure-based drug discovery, we screened millions of compounds against the NMR structure of the RGS12 RGS domain (Protein Data Bank id 2EBZ; state 2) to identify 96 candidate compounds, followed by experimental testing of these candidate compounds using the Transcreener® GDP RGScreen™ developed in partnership with BellBrook Labs [7,8]. Follow-up computational chemistry is being performed with Schrödinger's suite of molecular dynamics software. Results: Two hits out of the 96 candidate compounds were discovered to exhibit reproducible, double-digit micromolar IC50 values in the Transcreener® GDP RGScreen™ assay. We then tested 192 analogs of the two original hits and discovered 33 analogs with measurable IC50 values. Of the 33 congeneric compounds, all but one of the active congeners were structurally related to one of the original two hits, with a wide spread of IC50 values and many with improved potencies (IC50RGS12 = 0.84 – 153.2 microM). These hits do not inhibit the intrinsic GTPase activity of G-alpha. Conclusions: To increase diversity of the chemical scaffolds capable of inhibiting RGS12 function in vitro, we are now performing computational modeling of this initial set of 33 congeneric compounds, including CPU-based molecular docking and GPU-based shape screening of new chemical libraries (including those sourced from MilliporeSigma, MolPort, and Enamine) using Schrödinger algorithms on a local HPC cluster. In vitro binding and structural studies, cell-based studies, and pre-clinical animal studies are also being planned to further characterize these congeneric compounds and future divergent chemotypes. References: [1] https://www.ncbi.nlm.nih.gov/books/NBK424849/ [2] https://nida.nih.gov/publications/drugs-brains-behavior-science-addiction/drugs-brain [3] https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5942192/ [4] https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6785087/ [5] https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7992303/ [6] https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2084260/ [7] https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2795102/ [8] https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3306263/Item Molecular modeling and binding function of the RGS12 PDZ domain variant associated with familial bipolar disorder(2024-03-21) Agogo-Mawuli, Percy; Bosch, Dustin; Oestreich, Emily; Siderovski, DavidPurpose – Bipolar disorder (BD) is a highly heritable neuropsychiatric disease characterized by recurrent episodes of depression and mania. BD is considered one of the most vexing health disorders to medicate appropriately, yet affects ~2% of U.S. adults and is the most costly mental health condition for American health insurers nationwide [1]. In reporting the whole-exome sequencing of 81 individuals from 27 multiply-affected BD families, Forstner et al. [2] discovered a single nucleotide polymorphism co-segregating with BD in the gene encoding “Regulator of G protein Signaling” (RGS) type 12, predicted to cause a missense change (arginine-59 to glutamine) in the sequence of the encoded protein’s PDZ domain. RGS proteins are negative regulators of neurotransmitter signaling via G protein-coupled receptors (GPCRs) [3], specifically serving to accelerate signaling shutoff by increasing the GTP hydrolysis rate of GPCR-associated G-alpha subunits [4]. In prior mouse genetics studies, the Siderovski lab has demonstrated that RGS12 acts to regulate kappa opioid receptor signaling and extracellular dopamine levels in the basal ganglia of the brain, and that RGS12 is able to bind to various protein targets in neurons, such as the kinase MEK2 via its PDZ domain. To address whether the R59Q amino-acid variation in the RGS12 PDZ domain associated with BD affects the function of the RGS12 protein, molecular modeling and biochemical binding experiments were performed. Methods – Schrodinger’s software suite of molecular modeling and dynamics tools was employed to create structural models of the R59Q variant of RGS12’s PDZ domain, starting from the published NMR-derived structure of the domain with wildtype sequence (i.e., Protein Data Bank record id 2KV8). Desmond molecular dynamics software was used to relax both structural models and then assess their overall stability over 50 nanoseconds of elapsed simulation. Models of the wildtype and R59Q variant PDZ domain bound to short polypeptides from the C-termini of candidate RGS12-interacting proteins were similarly created and molecular dynamics simulations performed. Both PDZ domains were expressed as glutathione-S-transferase (GST) fusion proteins by E. coli cultures, purified by fast protein liquid chromatography (FPLC), and then tested for their binding to biotinylated polypeptides from the C-termini of candidate RGS12-interacting proteins using surface plasmon resonance (SPR). Results and Conclusion – The position of the missense change within RGS12 was not predicted to directly engage the polypeptide binding-site of the PDZ domain; however, when tested using SPR, the fusion protein bearing the R59Q-substituted PDZ domain exhibited less binding affinity for target polypeptide partners than the wildtype sequence. These initial in silico and in vitro findings suggest that the R59Q variant associated with BD may lead to reduced RGS12 protein function in vivo. References: [1] PMID: 35816713; DOI: 10.7326/AITC202207190 [2] PMID: 32066727; PMCID: PMC7026119; DOI: 10.1038/s41398-020-0732-y [3] PMID: 21737532; PMCID: PMC3141876; DOI: 10.1124/pr.110.003038 [4] PMID: 20351284; PMCID: PMC2872438; DOI: 10.1073/pnas.0912934107