Biology subjects

Lak, P.

Publications and source records attributed to Lak, P..

5 recordsLinked to original sources

bioRxiv2022

Reference compounds for characterizing cellular injury in high-content cellular morphology assays

Robust, generalizable approaches to identify compounds efficiently with undesirable mechanisms of action in complex cellular assays remain elusive. Such a process would be useful for hit triage during high-throughput screening and, ultimately, predictive toxicology during drug development. We generated cell painting and cellular health profiles for 218 prototypical cytotoxic and nuisance compounds in U-2 OS cells in a concentration-response format. A diversity of compounds causing cellular damage produced bioactive cell painting morphologies, including cytoskeletal poisons, genotoxins, nonspecific electrophiles, and redox-active compounds. Further, we show that lower quality lysine acetyltransferase inhibitors and nonspecific electrophiles can be distinguished from more selective counterparts. We propose that the purposeful inclusion of cytotoxic and nuisance reference compounds such as those profiled in this Resource will help with assay optimization and compound prioritization in complex cellular assays like cell painting.

pharmacology and toxicology↗

bioRxiv2022

Structure-based Discovery of Conformationally Selective Inhibitors of the Serotonin Transporter

The serotonin transporter (SERT) removes synaptic serotonin and is the target of anti-depressant drugs. SERT adopts three conformations: outward-open, occluded, and inward-open. All known inhibitors target the outward-open state except ibogaine, which has an unusual anti-depressant profile and stabilizes the inward-open conformation. Unfortunately, ibogaine is promiscuous and cardiotoxic, limiting understanding of inward-open state ligands. We computationally docked over 200 million small molecules against the ibogaine stabilized inward-open state of SERT. Thirty-six top-ranking compounds were synthesized and thirteen inhibited with potencies ranging from 29 to 5000 nM. Structure-based optimization led to two novel inhibitors with Ki values down to 3 nM. The new molecules stabilized an outward-closed state of the transporter and had little activity against off-targets. A cryo-EM structure of one of these bound to SERT confirmed the predicted geometry. In mouse behavioral assays, both had anxiolytic and anti-depressant activity, with potencies up to 200 better than fluoxetine.

biochemistry↗

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↗

bioRxiv2021

Energy Penalties Enhance Flexible Receptor Docking in a Model Cavity

Protein flexibility remains a major challenge in library docking due to difficulties in sampling conformational ensembles with accurate probabilities. Here we use the model cavity site of T4 Lysozyme L99A to test flexible receptor docking with energy penalties from molecular dynamics (MD) simulations. Crystallography with larger and smaller ligands indicates that this cavity can adopt three major conformations, open, intermediate, and closed. Since smaller ligands typically bind better to the cavity site, we anticipate an energy penalty for cavity opening. To estimate its magnitude, we calculate conformational preferences from MD simulations. We find that including a penalty term is essential for retrospective ligand enrichment, otherwise high-energy states dominate the docking. We then prospectively docked a library of over 900,000 compounds for new molecules binding to each conformational state. Absent a penalty term, the open conformation dominated the docking results; inclusion of this term led to a balanced sampling of ligands against each state. High ranked molecules were experimentally tested by Tm-upshift and X-ray crystallography. From 33 selected molecules, we identified 18 new ligands and determined 13 crystal structures. Most interesting were those bound to the open cavity, where the buried site opens to bulk solvent. Here, highly unusual ligands for this cavity had been predicted, including large ligands with polar tails; these were confirmed both by binding and by crystallography. In docking, incorporating protein flexibility with thermodynamic weightings may thus access new ligand chemotypes. The MD approach to accessing and, crucially, weighting such alternative states may find general applicability. Significance StatementThe dynamic nature of biomolecules is typically neglected in docking screens for ligand discovery. Key to benefitting from various receptor conformations is not only structural but also thermodynamic information. Here we test a general approach that uses conformational preferences from enhanced and conventional MD simulations to account for the cost of transitions to high energy states. Including this information as a conformational penalty term in a docking scoring function, we perform retrospective and prospective screens and experimentally confirm novel ligands with Tm-upshift and X-ray crystallography.

biophysics↗

bioRxiv2018

Conserved tau microtubule-binding repeat histidines confer pH-dependent tau-microtubule association

Tau, a member of the MAP2/tau family of microtubule-associated proteins, functions to stabilize and organize axonal microtubules in healthy neurons. In contrast, tau dissociates from microtubules and forms neurotoxic extracellular aggregates in neurodegenerative tauopathies. MAP2/tau family proteins are characterized by three to five conserved, intrinsically disordered repeat regions that mediate electrostatic interactions with the microtubule surface. We use molecular dynamics, microtubule-binding experiments and live cell microscopy to show that highly conserved histidine residues near the C terminus of each MT-binding repeat are pH sensors that can modulate tau-MT interaction strength within the physiological intracellular pH range. At lower pH, these histidines are positively charged and form cation-{pi} interactions with phenylalanine residues in a hydrophobic cleft between adjacent tubulin dimers. At higher pH, tau deprotonation decreases microtubule-binding both in vitro and in cells. However, electrostatic and hydrophobic characteristics of histidine are required for tau-MT-binding as substitution with constitutively positively charged, non-aromatic lysine or uncharged alanine greatly reduces or abolishes tau-MT binding. Consistent with these findings, tau-MT binding is reduced in a cancer cell model with increased intracellular pH but is rapidly rescued by decreasing pH to normal levels. Thus, these data add a new dimension to the intracellular regulation of tau activity and could be relevant in normal and pathological conditions.

cell biology↗