Biology subjects

Bisignano, P.

Publications and source records attributed to Bisignano, P..

5 recordsLinked to original sources

bioRxiv2025

A conserved mechanism of LRRC8 channel inhibition by distinct drugs

We employed a Leucine Rich Repeat Containing 8 (LRRC8) channel chimera, termed 8C-8A(IL125), to investigate the molecular mechanism of action of the novel volume-sensitive anion channel (VRAC) inhibitor, zafirlukast. 8C-8A(IL125) comprises LRRC8C (8C) and 25 residues from LRRC8A (8A) intracellular loop 1 (IL1) and forms volume-sensitive, structurally defined heptameric channels with normal pharmacological properties. In silico docking and modeling with AlphaFold3 identified a putative zafirlukast binding site comprising the amino (N)-terminal domain (NTD) and inter-subunit fenestrae between transmembrane (TM) helices 1 and 2. Consistent with this model, mutations in NTD, TM1, and TM2 alter 8C-8A(IL125) and heteromeric 8A/8C sensitivity to zafirlukast and the structurally distinct drug pranlukast. Inhibition is not mediated by extracellular pore block or the so-called lipid gate. Mutations or low pH conditions that enhance voltage-dependent inactivation also increase zafirlukast sensitivity. We propose zafirlukast and pranlukast promote channel inactivation through destabilization of the pore.

pharmacology and toxicology↗

bioRxiv2025

Lipid-mediated mechanism of drug extrusion by a heterodimeric ABC exporter

Multidrug transport by ATP binding cassette (ABC) exporters entails a mechanism to modulate drug affinity across the transport cycle. Here, we combine cryo-EM and molecular dynamics (MD) simulations to illuminate how lipid competition modulates substrate affinity to drive its translocation by ABC exporters. We determined cryo-EM structures of the ABC transporter BmrCD in drug-loaded inward-facing (IF) and outward-facing (OF) conformations in lipid nanodiscs to reveal the structural basis of alternating access, details of drug-transporter interactions, and the scale of drug movement between the two conformations. Remarkably, the structures uncovered lipid molecules bound in or near the transporter vestibule along with the drugs. MD trajectories from the IF structure show that these lipids stimulate drug disorder and translocation towards the vestibule apex. Similarly, bound lipids enter the OF vestibule and weaken drug-transporter interactions facilitating drug release. Our results complete a near-atomic model of BmrCDs conformational cycle and advance a general mechanism of lipid-driven drug transport by ABC exporters.

biophysics↗

bioRxiv2022

The cilia enriched oxysterol 7β,27-DHC is required for polycystin activation

PC-1 and PC-2 form a heteromeric ion channel complex (hereafter called the Polycystin complex) that is abundantly expressed on primary cilia of renal epithelial cells. Mutations within the polycystin complex cause Autosomal Dominant Polycystic Kidney Disease (ADPKD). The Polycystin complex forms a non-selective cation channel, yet the spatial and temporal regulation of the polycystin complex within the ciliary membrane remains poorly understood, partially due to technical limitations posed by the tiny ciliary compartment. Here, we employ our novel assays to functionally reconstitute the polycystin complex in the plasma membrane. Using whole-cell and ciliary patch-clamp recordings we identified a ciliary enriched oxysterol, 7{beta},27-DHC, as a critical component required for activation of the polycystin complex. We identified a novel oxysterol binding pocket in PC-2 using molecular docking simulation. We also identified two amino acids within the PC-2 oxysterol binding pocket, E208 and R581, to be critical for 7{beta},27-DHC dependent polycystin activation in both the plasma membrane and ciliary compartment. Further, we can show that the pharmacological and genetic inhibition of oxysterol synthesis by carbenoxolone (CNX) reduces channel activity in primary cilia. Our findings identified a unique second messenger that regulates the polycystin complex. We hypothesize that cilia-enriched lipids license the polycystin complex to be functional only in the ciliary organelle, thus providing novel insights into the spatial regulation of the polycystin complex. Our results also establish a framework to target the same allosteric regulatory site in the polycystin complex to identify activators of the polycystin channels as novel therapeutic strategies for ADPKD.

physiology↗

bioRxiv2020

Selective Targeting of Ras Mutant Cancers via a New Small Molecule

Mutations in the Ras family of oncogenes are implicated in 33% of human cancers, making Ras an intensely pursued target in drug discovery. As an alternative to direct pharmacological inhibition of Ras, we looked for sensitivities in RAS mutant cells. Using a small molecule screen in cell lines with mutations in Ras and its effector Raf, we discovered 249C as a Ras-mutant selective cytotoxic agent against a spectrum of RAS-mutant cancers. By combining CRISPR chemical-genetic screening, comparative profiling and chemoproteomics, we identified that 249C binds to a unique subunit on vacuolar (V)-ATPase with nanomolar affinity, inhibiting its biochemical activity and, unexpectedly, altering V-ATPase translocation in Ras-induced macropinocytosis. Via binding to V-ATPase, 249C prevents lysosomal acidification and inhibits autophagy and macropinocytosis pathways that several Ras-driven cancers rely on for survival. In characterizing 249Cs mechanism, we show that potency varies with the identity of the RAS driver mutation highlighting a mutant-specific dependence on autophagy and macropinocytosis. Indeed, 249C potently inhibits tumor growth without adverse side effects in a mouse xenograft model of KRAS-driven non-small cell lung cancer. These data establish proof-of-concept for targeting V-ATPase as a way to indirectly target specific Ras mutants, and provide a fundamental link between V-ATPase localization and specific Ras mutant tumor-related activity.

cancer biology↗

bioRxiv2019

What is that molecular machine really doing? Automated exploration of alternative transporter mechanisms

Motivated by growing evidence for pathway heterogeneity and alternative functions of molecular machines, we demonstrate a computational approach for investigating two questions: (1) Are there multiple mechanisms (state-space pathways) by which a machine can perform a given function, such as cotransport across a membrane? (2) How can additional functionality, such as proofreading/error-correction, be built into machine function using standard biochemical processes? Answers to these questions will aid both the understanding of molecular-scale cell biology and the design of synthetic machines. Focusing on transport in this initial study, we sample a variety of mechanisms by employing Metropolis Markov chain Monte Carlo. Trial moves adjust transition rates among an automatically generated set of conformational and binding states while maintaining fidelity to thermodynamic principles and a user-supplied fitness/functionality goal. Each accepted move generates a new model. The simulations yield both single and mixed reaction pathways for cotransport in a simple environment with a single substrate along with a driving ion. In a "competitive" environment including an additional decoy substrate, several qualitatively distinct reaction pathways are found which are capable of extremely high discrimination coupled to a leak of the driving ion, akin to proofreading. The array of functional models would be difficult to find by intuition alone in the complex state-spaces of interest. Author summaryMolecular machines, which operate on the nanoscale, are proteins/complexes that perform remarkable tasks such as the selective absorption of nutrients into the cell by transporters. These complex machines are often described using a fairly simple set of states and transitions that may not account for the stochasticity and heterogeneity generally expected at the nanoscale at body temperature. New tools are needed to study the full array of possibilities. This study presents a novel in silico method to systematically generate testable molecular-machine kinetic models and explore alternative mechanisms, applied first to membrane transport proteins. Our initial results suggest these transport machines may contain mechanisms which detoxify the cell of an unwanted toxin, as well as significantly discriminate against the import of the toxin. This novel approach should aid the experimental study of key physiological processes such as renal glucose re-absorption, rational drug design, and potentially the development of synthetic machines.

biophysics↗