Biology subjects

Powers, A. S.

Publications and source records attributed to Powers, A. S..

8 recordsLinked to original sources

bioRxiv2026

Mechanism of Gating and Isoform-Specific Inhibition in Renal CLC Chloride Channels

Hyponatremia is a prevalent disorder marked by excess water retention and substantial morbidity, motivating interest in the CLC-Ka chloride channel as a therapeutic target. Selectively inhibiting CLC-Ka without affecting the closely related CLC-Kb is essential for preventing serious side effects. However, developing isoform-selective inhibitors has been challenging because most small molecules do not distinguish between CLC-Ka and CLC-Kb, and the basis for selectivity in the few known exceptions remains unclear. The small molecule BIM1 preferentially inhibits CLC-Ka over CLC-Kb, providing an opportunity to dissect isoform-specific pharmacology. To investigate this mechanism, we determined cryo-EM structures of BIM1 and BIM15, a related nonselective analog, bound to a CLC-K variant engineered to match the human CLC-Ka binding pocket. Structural and computational analyses reveal that inhibition and isoform selectivity are anchored by interactions with a conserved lysine, with surrounding binding-site residues subtly tuning the local electrostatic environment to promote or disfavor these contacts. These analyses further identify a dynamic extracellular loop that intermittently occludes the shared pathway accessing the inhibitor-binding site and pore. Bound BIM15 engages this gating loop more extensively than BIM1, suggesting that differential loop engagement contributes to inhibitor selectivity, a prediction validated by mutagenesis. Because loop dynamics block the pore, we examined the structural impact of Ca{superscript 2}, which favors opening, and found the gating loop ordered and withdrawn from the pathway. Together, these findings define how binding-site microenvironments and gating-loop dynamics shape isoform-specific inhibition and pore access in CLC-K channels. Significance StatementHyponatremia is a major clinical problem with limited therapeutic options. The kidney chloride channel CLC-Ka is an attractive drug target, but its high sequence identity to CLC-Kb has hindered the development of isoform-selective inhibitors needed for safe therapy. A low-micromolar CLC-Ka-selective inhibitor had been identified, providing a foothold for drug development, but the structural basis of its selectivity was unknown. Here, by integrating cryo-EM structures with molecular dynamics simulations, we define the inhibitor-binding site and reveal the mechanism that enables preferential CLC-Ka inhibition. We further show that a dynamic extracellular loop functions as a gating element shaping inhibitor access and engagement. These findings establish a mechanistic foundation for developing improved treatments for hyponatremia.

biophysics↗

bioRxiv2025

MedSAGE: Bridging Generative AI and Medicinal Chemistry for Structure-Based Design of Small Molecule Drugs

While generative AI is transforming the de novo design of proteins, its effectiveness for structure-based design of small molecules remains limited. Current methods, including diffusion models, often produce small molecules with difficult-to-synthesize structures, poor medicinal chemistry properties, and limited target selectivity. To address these limitations, we introduce MedSAGE, a novel generative AI framework that adapts diffusion models specifically for de novo small-molecule design. Rather than using atoms or strings, we develop a novel representation for generating molecules using fragments relevant for medicinal chemistry. Chemical and geometric information characterizing these fragments is embedded in a smooth and interpretable latent space. We also develop an algorithm to optimize the connectivity between generated fragments while preserving chemical validity and synthesizability. In a benchmark of multiple methods across 25 therapeutically relevant protein targets, MedSAGE achieved state-of-the-art performance, producing synthesizable, drug-like molecules with predicted affinity and selectivity closely matching known drugs and drug candidates. Compared to large-scale virtual screening, MedSAGE produced molecules with high predicted affinity over 100 times more efficiently. Our results demonstrate that MedSAGE is already practically useful and paves the way for next-generation tools in structure-guided drug design.

biophysics↗

bioRxiv2023

CryoEM structures of the human CLC-2 voltage gated chloride channel reveal a ball and chain gating mechanism

CLC-2 is a voltage-gated chloride channel that contributes to electrical excitability and ion homeostasis in many different mammalian tissues and cell types. Among the nine mammalian CLC homologs, CLC-2 is uniquely activated by hyperpolarization, rather than depolarization, of the plasma membrane. The molecular basis for the divergence in polarity of voltage gating mechanisms among closely related CLC homologs has been a long-standing mystery, in part because few CLC channel structures are available, and those that exist exhibit high conformational similarity. Here, we report cryoEM structures of human CLC-2 at 2.46 - 2.76 [A], in the presence and absence of the potent and selective inhibitor AK-42. AK-42 binds within the extracellular entryway of the Cl--permeation pathway, occupying a pocket previously proposed through computational docking studies. In the apo structure, we observed two distinct apo conformations of CLC-2 involving rotation of one of the cytoplasmic C-terminal domains (CTDs). In the absence of CTD rotation, an intracellular N-terminal 15-residue hairpin peptide nestles against the TM domain to physically occlude the Cl--permeation pathway from the intracellular side. This peptide is highly conserved among species variants of CLC-2 but is not present in any other CLC homologs. Previous studies suggested that the N-terminal domain of CLC-2 influences channel properties via a "ball-and-chain" gating mechanism, but conflicting data cast doubt on such a mechanism, and thus the structure of the N-terminal domain and its interaction with the channel has been uncertain. Through electrophysiological studies of an N-terminal deletion mutant lacking the 15-residue hairpin peptide, we show that loss of this short sequence increases the magnitude and decreases the rectification of CLC-2 currents expressed in mammalian cells. Furthermore, we show that with repetitive hyperpolarization WT CLC-2 currents increase in resemblance to the hairpin-deleted CLC-2 currents. These functional results combined with our structural data support a model in which the N-terminal hairpin of CLC-2 stabilizes a closed state of the channel by blocking the cytoplasmic Cl--permeation pathway.

biophysics↗

bioRxiv2023

A non-canonical mechanism of GPCR activation

The goal of designing safer, more effective drugs has led to tremendous interest in molecular mechanisms through which ligands can precisely manipulate signaling of G-protein-coupled receptors (GPCRs), the largest class of drug targets. Decades of research have led to the widely accepted view that all agonists--ligands that trigger GPCR activation--function by causing rearrangement of the GPCRs transmembrane helices, opening an intracellular pocket for binding of transducer proteins. Here we demonstrate that certain agonists instead trigger activation of free fatty acid receptor 1 by directly rearranging an intracellular loop that interacts with transducers. We validate the predictions of our atomic-level simulations by targeted mutagenesis; specific mutations which disrupt interactions with the intracellular loop convert these agonists into inverse agonists. Further analysis suggests that allosteric ligands could regulate signaling of many other GPCRs via a similar mechanism, offering rich possibilities for precise control of pharmaceutically important targets.

molecular biology↗

bioRxiv2022

Modulation of cannabinoid receptor signaling by endocannabinoids

Endocannabinoids (eCBs) are endogenous lipid molecules that activate the cannabinoid receptor 1 (CB1), a G protein coupled receptor (GPCR) that signals primarily through the Gi/o family of G proteins to regulate neurotransmitter release. Consequently, CB1 is an important therapeutic target for several neurological disorders. How eCBs interact with CB1 is not known and the downstream signaling they activate is not well understood. In this study we show that eCBs do not activate Gi1 as much as synthetic cannabinoids. To characterize activation of CB1 by eCB, we formed an eCB analogue-bound (AMG315) CB1-Gi signaling complex for structural studies. The structure reveals differences in the orthosteric ligand binding pocket not seen in the previous CB1 structures, providing insights into the structural determinants of ligand efficacy. In combination with signaling and simulation data, this study provides mechanistic insights into CB1 activation by different classes of ligands, and sheds light on the G protein preferences between endogenous and exogenous ligands.

biochemistry↗

bioRxiv2020

Leveraging non-structural data to predict structures of protein-ligand complexes

Over the past fifty years, tremendous effort has been devoted to computational methods for predicting properties of ligands that bind macromolecular targets, a problem critical to rational drug design. Such methods generally fall into two categories: physics-based methods, which directly model ligand interactions with the target given the targets three-dimensional (3D) structure, and ligand-based methods, which predict ligand properties given experimental measurements for similar ligands. Here we present a rigorous statistical framework to combine these two sources of information. We develop a method to predict a ligands pose--the 3D structure of the ligand bound to its protein target--that leverages a widely available source of information: a list of other ligands that are known to bind the same target but for which no 3D structure is available. This combination of physics-based and ligand-based modeling improves upon state-of-the-art pose prediction accuracy across all major families of drug targets. As an illustrative application, we predict binding poses of antipsychotics and validate the results experimentally. Our statistical framework and results suggest broad opportunities to predict diverse ligand properties using machine learning methods that draw on physical modeling and ligand data simultaneously.

biophysics↗

bioRxiv2020

Structure of human ferroportin reveals molecular basis of iron homeostasis

The serum iron level in humans is tightly controlled by the action of the hormone hepcidin on the iron efflux transporter ferroportin. Hepcidin negatively regulates iron absorption and recycling by inducing ferroportin internalization and degradation. Aberrant ferroportin activity can lead to diseases of iron overload, like hemochromatosis, or iron limitation anemias. Here, we determined cryogenic electron microscopy (cryo-EM) structures of ferroportin in lipid nanodiscs, both in the apo state and in complex with cobalt, an iron mimetic, and hepcidin. These structures and accompanying molecular dynamics simulations identify two divalent metal binding sites within the N- and C-domains of ferroportin. Hepcidin binds ferroportin in an outward-open conformation and completely occludes the iron efflux pathway. The carboxy-terminus of hepcidin directly contacts the divalent metal in the FPN C-domain. We further show that hepcidin binding to ferroportin is coupled to iron binding, with an 80-fold increase in hepcidin affinity in the presence of iron. These results suggest a new model for hepcidin regulation of ferroportin, where only iron loaded ferroportin molecules are targeted for degradation. More broadly, our structural and functional insights are likely to enable more targeted manipulation of the hepcidin-ferroportin axis in disorders of iron homeostasis.

biochemistry↗

bioRxiv2019

Assessing positioning in enzymatic catalysis via ketosteroid isomerase conformational ensembles

How enzymes achieve their enormous rate enhancements remains a central question in biology, and our understanding to date has impacted drug development, influenced enzyme design, and deepened our appreciation of evolutionary processes. While enzymes position catalytic and reactant groups in active sites, physics requires that atoms undergo constant motion. Numerous proposals have invoked positioning or motions as central for enzyme function, but a scarcity of experimental data has limited our understanding of positioning and motion, their relative importance, and their changes through the enzymes reaction cycle. To examine positioning and motions and test catalytic proposals, we collected "room temperature" X-ray crystallography data for P. putida ketosteroid isomerase (KSI), and we obtained conformational ensembles for this and a homologous KSI from multiple PDB crystal structures. Ensemble analyses indicated limited change through KSIs reaction cycle. Active site positioning was on the 1-1.5 [A] scale, and was not exceptional compared to non-catalytic groups. The KSI ensembles provided evidence against catalytic proposals invoking oxyanion hole geometric discrimination between the ground state and transition state or highly precise general base positioning. Instead, increasing or decreasing positioning of KSIs general base reduced catalysis, suggesting optimized [A]ngstrom-scale conformational heterogeneity that allows KSI to efficiently catalyze multiple reaction steps. Ensemble analyses of surrounding groups for WT and mutant KSIs provided insights into the forces and interactions that allow and limit active site motions. Most generally, this ensemble perspective extends traditional structure-function relationships, providing the basis for a new era of "ensemble-function" interrogation of enzymes.

biophysics↗