Biology subjects

Kaplan, A. L.

Publications and source records attributed to Kaplan, A. L..

4 recordsLinked to original sources

bioRxiv2024

Regulation of transposons within medium spiny neurons enables molecular and behavioral responses to cocaine

A more complete understanding of the molecular mechanisms by which substance use is encoded in the brain could illuminate novel strategies to treat substance use disorders, including cocaine use disorder (CUD). We have previously discovered that Zfp189, which encodes a Kruppel-associated box zinc finger protein (KZFP) transcription factor (TF), differentially accumulates in nucleus accumbens (NAc) Drd1+ and Drd2+ medium spiny neurons (MSNs) over the course of cocaine exposure and is causal in producing MSN functional and behavioral changes to cocaine1. Here, we aimed to illuminate the brain cell-type specific molecular mechanisms through which this KZFP TF produces CUD-related brain changes, with emphasis on investigating transposable elements (TEs), since KZFPs like ZFP189 are known regulators of TEs2-6. First, we annotated TEs in existing single nuclei RNA-sequencing (snRNAseq) datasets of rodents that were exposed to either acute or repeated cocaine. We discovered that expression of NAc TEs was dramatically altered by cocaine experience, the most sensitive NAc cell-type was MSNs, and TEs in Drd1+ MSNs were considerably more dynamic over the course of cocaine exposure than TEs in Drd2+ MSNs. To determine the causality of this TE dysregulation within NAc MSNs in cocaine-induced brain changes, we virally delivered conditional synthetic ZFP189 TFs of our own design to Drd1+ or Drd2+ MSNs. These synthetic ZFP189 TFs are capable of directly activating (ZFP189VPR) or repressing (ZFP189WT) brain TEs2. We discover that behavioral and cell morphological adaptations to cocaine are produced by activating TEs with ZFP189VPR in Drd1+ MSNs or stabilizing TEs with ZFP189WT in Drd2+ MSNs, revealing a persistent opponent process balanced across MSN subtypes and weighted by TE stability and consequent gene expression within MSN subtype. We next performed snRNAseq of the whole NAc virally manipulated with ZFP189 TFs. We observed that, relative to ZFP189WT, NAc manipulated with ZFP189VPR impeded cocaine-induced gene expression in NAc cell-types, including both Drd1+ and Drd2+ MSNs. Within either MSN subtype, the consequence of normal ZFP189 function was to enhance immune-related gene expression, and ZFP189VPR impeded these gene expression profiles. We finally performed cocaine intravenous self-administration to determine the consequence of NAc ZFP189-mediated transcriptional control on cocaine use behaviors. We observed that ZFP189VPR impeded any increases in active lever responses following a period forced cocaine abstinence. This research demonstrates that KZFP-mediated transcriptional repression of TEs within NAc MSNs is a causal molecular step in enabling gene expression and subsequent cellular and behavioral responses to cocaine use, and the use of ZFP189VPR in this work demonstrates cell-type specific mechanistic strategies to block CUD-related brain adaptations, which may inform future CUD treatments.

neuroscience↗

bioRxiv2023

Structure-based discovery of positive allosteric modulators for the calcium sensing receptor

Drugs acting as positive allosteric modulators (PAMs) to enhance the activation of the calcium sensing receptor (CaSR) and to suppress parathyroid hormone (PTH) secretion can treat hyperparathyroidism but suffer from side effects including hypocalcemia and arrhythmias. Seeking new CaSR modulators, we docked libraries of 2.7 million and 1.2 billion molecules against transforming pockets in the active-state receptor dimer structure. Consistent with simulations suggesting that docking improves with library size, billion-molecule docking found new PAMs with a hit rate that was 2.7-fold higher than the million-molecule library and with hits up to 37-fold more potent. Structure-based optimization of ligands from both campaigns led to nanomolar leads, one of which was advanced to animal testing. This PAM displays 100-fold the potency of the standard of care, cinacalcet, in ex vivo organ assays, and reduces serum PTH levels in mice by up to 80% without the hypocalcemia typical of CaSR drugs. Cryo-EM structures with the new PAMs show that they induce residue rearrangements in the binding pockets and promote CaSR dimer conformations that are closer to the G-protein coupled state compared to established drugs. These findings highlight the promise of large library docking for therapeutic leads, especially when combined with experimental structure determination and mechanism. One sentence summaryStructure-based virtual screening uncovers novel CaSR allosteric modulators with enhanced efficacy and less side effects.

biophysics↗

bioRxiv2023

Structure-based discovery of CFTR potentiators and inhibitors

The cystic fibrosis transmembrane conductance regulator (CFTR) is a crucial ion channel whose loss of function leads to cystic fibrosis, while its hyperactivation leads to secretory diarrhea. Small molecules that improve CFTR folding (correctors) or function (potentiators) are clinically available. However, the only potentiator, ivacaftor, has suboptimal pharmacokinetics and inhibitors have yet to be clinically developed. Here we combine molecular docking, electrophysiology, cryo-EM, and medicinal chemistry to identify novel CFTR modulators. We docked [~]155 million molecules into the potentiator site on CFTR, synthesized 53 test ligands, and used structure-based optimization to identify candidate modulators. This approach uncovered novel mid-nanomolar potentiators as well as inhibitors that bind to the same allosteric site. These molecules represent potential leads for the development of more effective drugs for cystic fibrosis and secretory diarrhea, demonstrating the feasibility of large-scale docking for ion channel drug discovery.

biophysics↗

bioRxiv2021

Structure-based design of a chemical probe set for the 5-HT5A serotonin receptor

The 5-HT5A receptor (5-HT5AR), for which no selective agonists and only a few antagonists exist, remains the least understood serotonin (5-HT) receptor. A single commercial antagonist (SB-699551) has been widely used to investigate central nervous system (CNS) 5-HT5AR function in neurological disorders, including pain. However, because SB-699551 has affinity for many 5-HTRs, lacks inactive property-matched controls, and has assay interference concerns, it has liabilities as a chemical probe. To better illuminate 5-HT5AR function, we developed a probe set through iterative rounds of molecular docking, pharmacological testing, and optimization. Docking over six million lead-like molecules against a 5-HT5AR homology model identified five mid-M ligand starting points with unique scaffolds. Over multiple rounds of structure-based design and testing, a new quinoline scaffold with high affinity and enhanced selectivity for the 5-HT5AR was developed, leading to UCSF678, a 42 nM arrestin-biased partial agonist at the 5-HT5AR with a much more restricted off-target profile and decreased assay liabilities vs. SB-699551. Site-directed mutagenesis supported the docked pose of UCSF678, which was also consistent with recent published 5-HTR structures. Surprisingly, property-matched analogs of UCSF678 that were either inactive across 5-HTRs or retained affinity for UCSF678s off-targets revealed that 5-HT5AR engagement is nonessential for alleviating pain in a mouse model, contrary to previous studies using less-selective ligands. Relative to SB-699551, these molecules constitute a well-characterized and more selective probe set with which to study the function of the 5-HT5A receptor. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/471449v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@8fb24dorg.highwire.dtl.DTLVardef@2dd84org.highwire.dtl.DTLVardef@1d3346org.highwire.dtl.DTLVardef@1117331_HPS_FORMAT_FIGEXP M_FIG Table of Contents Graphic C_FIG

biophysics↗