Small molecule regulation of endogenous transcription factors for circuit-specific neuromodulation

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PROJECT SUMMARY Methods for regulating cellular processes within distinct populations of neurons are needed to elucidate relationships between molecular mechanisms, circuits, and behavior; and to develop cell type- or circuit- selective treatments for neurological disorders. We propose a novel, non-genetic, small molecule method— transcription factor-chemically induced proximity (TF-CiP)—that harnesses the cell type- and circuit-specificity of endogenous transcription factors to regulate gene expression in subsets of neurons. TF-CiP utilizes a bifunctional small molecule to heterodimerize an “anchor” transcription factor, which naturally binds to a target gene, with a “hijacked” transcription factor, which enhances or represses transcription of the target gene. Cell specificity is determined from the intersection of expression of each transcription factor. TF-CiP can theoretically be adapted for any organism and because transcription factors are well conserved, it is possible that the same TF-CiP small molecule can be used to modulate neuronal processes across animal species. We will develop TF-CiP to regulate the expression of the rate-limiting enzyme for brain serotonin synthesis, TPH2, as a means to tune serotonin levels in subsets of serotonergic neurons. Although the population of central serotonergic neurons is relatively small, these neurons send projections throughout the brain and serve important roles in regulating mood, anxiety, sleep, and social behavior. Achieving circuit-specificity for a serotonin-modulatory small molecule would be an improvement over current therapies, which can have undesirable side-effects due to indiscriminate targeting of serotonin signaling in the central and peripheral nervous systems. TPH2 transcription is regulated by stress, sex hormones, and several transcription factors. We leverage this knowledge along with single-cell RNA-sequencing and projection mapping data in serotonergic neurons as a resource for candidate TF-CiP transcription factors. In Aim 1, we will screen for transcription factors that regulate TPH2 transcription cooperatively upon chemically-induced heterodimerization of their FRB and FKBP tags. This screen will employ a Tph2-Venus reporter and orthophthalaldehyde- mediated serotonin visualization as readouts in serotonergic cells. In Aim 2, we will work with structural biologists and chemists to synthesize bifunctional TF-CiP molecules for TPH2 transcriptional regulation. This will involve virtual binding screens of over 8 million small molecules, binding validation using surface plasmon resonance and isothermal titration calorimetry, small molecule functionalization, and chemical linkage of two transcription factor-binding molecules. In Aim 3, TF-CiP molecules will be screened for cytotoxicity, selectivity, and efficacy at regulating TPH2 transcription and serotonin synthesis in cells. In Aim 4, we will test TF-CiP molecules for efficacy and selectivity in vivo using murine social behavior assays and brain immunostaining, respectively. Successful completion of this study will provide the first neuropharmaceutic that is capable of targeting specific subsets of neurons or circuits without the aid of exogenous genetically-encoded proteins.