Biology subjects

Choudhury, K.

Publications and source records attributed to Choudhury, K..

4 recordsLinked to original sources

bioRxiv2021

Differential Binding Kinetics for Evaluating Immune Checkpoint Inhibitors in Serum

The ability to characterize the binding kinetics of drug-target interactions in a biologically relevant matrix, such as serum or plasma, remains a fundamental challenge in drug discovery. We apply a novel label-based giant magnetoresistance (GMR) biosensor platform to measure protein binding kinetics and affinities of drug-target pairs in buffer and different levels of serum. Specifically, we evaluate three well-established immune checkpoint inhibitors, pembrolizumab, nivolumab and atezolizumab and compare the results with label-free kinetic platforms: surface plasmon resonance (SPR) and bio-layer interferometry (BLI). Labeling of analytes does not affect their association and dissociation rates (on and off rates) from GMR biosensors which enables kinetic measurements in biologically relevant matrices. Only the GMR biossensors is consistently suitable for measuring binding kinetics in up to 80% serum. The faster and different off-rates of the three immune checkpoint inhibitors in the presence of serum should be considered when modeling their pharmacological performance. TeaserWe reveal the effects of serum on binding kinetics of antibody drugs, relevant to the pharmacological performance of immunotherapeutic.

bioengineering↗

bioRxiv2021

An open state of a voltage-gated sodium channel involving a π-helix and conserved pore-facing Asparagine

Voltage-gated sodium (Nav) channels play critical roles in propagating action potentials and otherwise manipulating ionic gradients in excitable cells. These channels open in response to membrane depolarization, selectively permeating sodium ions until rapidly inactivating. Structural characterization of the gating cycle in this channel family has proved challenging, particularly due to the transient nature of the open state. A structure from the bacterium Magnetococcus marinus Nav (NavMs) was initially proposed to be open, based on its pore diameter and voltage-sensor conformation. However, the functional annotation of this model, and the structural details of the open state, remain disputed. In this work, we used molecular modeling and simulations to test possible open-state models of NavMs. The full-length experimental structure, termed here the -model, was consistently dehydrated at the activation gate, indicating an inability to conduct ions. Based on a spontaneous transition observed in extended simulations, and sequence/structure comparison to other Nav channels, we built an alternative {pi}-model featuring a helix transition and the rotation of a conserved asparagine residue into the activation gate. Pore hydration, ion permeation and state-dependent drug binding in this model were consistent with an open functional state. This work thus offers both a functional annotation of the full-length NavMS structure, and a detailed model for a stable Nav open state, with potential conservation in diverse ion-channel families.

biophysics↗

bioRxiv2020

Functional crosstalk between phosphorylation and disease-causing mutations in the cardiac sodium channel Nav1.5

The voltage-gated sodium channel Nav1.5 initiates the cardiac action potential. Alterations of its activation and inactivation properties due to mutations can cause severe, life-threatening arrhythmias. Yet despite intensive research efforts, many functional aspects of this cardiac channel remain poorly understood. For instance, Nav1.5 undergoes extensive post-translational modification in vivo, but the functional significance of these modifications is largely unexplored, especially under pathological conditions. This is because most conventional approaches are unable to insert metabolically stable post-translational modification mimics, thus preventing a precise elucidation of the contribution by these modifications to channel function. Here, we overcome this limitation by using protein semi-synthesis of Nav1.5 in live cells and carry out complementary molecular dynamics simulations. We introduce metabolically stable phosphorylation mimics on both WT and two pathogenic long-QT mutant channel backgrounds and decipher functional and pharmacological effects with unique precision. We elucidate the mechanism by which phosphorylation of Y1495 impairs steady-state inactivation in WT Nav1.5. Surprisingly, we find that while the Q1476R patient mutation does not affect inactivation on its own, it enhances the impairment of steady-state inactivation caused by phosphorylation of Y1495 through enhanced unbinding of the inactivation particle. We also show that both phosphorylation and patient mutations can impact Nav1.5 sensitivity towards the clinically used anti-arrhythmic drugs quinidine and ranolazine, but not flecainide. The data highlight that functional effects of Nav1.5 phosphorylation can be dramatically amplified by patient mutations. Our work is thus likely to have implications for the interpretation of mutational phenotypes and the design of future drug regimens. Significance statementThe cardiac sodium channel (Nav1.5) is crucial for generating a regular heartbeat. It is thus not surprising that mutations in its sequence have been linked to life-threatening arrhythmias. Interestingly, Nav1.5 activity can also be altered by posttranslational modifications, such as tyrosine phosphorylation. Our combination of protein engineering and molecular modeling studies has revealed that the detrimental effect of a long QT3 patient mutation is only exposed when a proximal tyrosine is phosphorylated. This suggests a dynamic crosstalk between the genetic mutation and a neighboring phosphorylation, a phenomenon that could be important in other classes of proteins. Additionally, we show that phosphorylation can affect the channels sensitivity towards clinically-relevant drugs, a finding that may prove important when devising patient-specific treatment plans.

biophysics↗

bioRxiv2020

Mechanism and effects of the skeletal muscle Nav1.4 inhibition by cannabidiol

ABSTRACTCannabis sativa contains active constituents called phytocannabinoids. Some phytocannabinoids are psychotropic and others are not. The primary non-psychotropic phytocannabinoid is cannabidiol (CBD), which is proposed to be therapeutic against many conditions, including muscle spasms. Mechanisms have been proposed for the action of CBD on different systems, involving multiple targets, including the voltage-gated sodium channel (Nav) family, which are heavily implicated in many of the conditions CBD has been reported to relieve. In this study, we investigated the modulatory mechanism of CBD on Nav1.4. Based on previous results, we tested the hypothesis that CBD mechanism of action involves: 1) modulation of membrane elasticity, which indirectly contributes to Nav inhibition; and 2) physical block of the Nav pore. We first performed molecular dynamic (MD) simulations to visualize CBD effects and localization inside the membrane, and then performed NMR to verify the MD results, showing CBD localizes below membrane headgroups. Then, we performed a gramicidin-based fluorescence (GFA) assay that showed CBD alters membrane elasticity. Next, we used site-directed mutagenesis in (F1586A) and around (WWWW) the Nav1.4 pore. Removing the local anesthetic binding site with F1586A reduced CBD block of INa. Occluding the fenestrations with WWWW blocked CBD access from the membrane into the Nav1.4 pore. However, stabilization of inactivation, via CBD-induced changes in membrane elasticity persisted, in WWWW. To investigate the potential therapeutic value of CBD against some Nav1.4 channelopathies, we used a pathogenic variant of Nav1.4, P1158S, known to cause myotonia and periodic paralysis. We found CBD reduces excitability in both wild-type and the mixed myotonia/periodic paralysis variant. Our in-vitro/in-silico results suggest that CBD may have therapeutic value against myotonia. Because Nav1.4 is crucial to skeletal muscle contraction, we used rat diaphragm myography and found the presence of saturating levels of CBD reduces skeletal muscle contraction.SUMMARY We used multidisciplinary approaches to show the mechanism and pathway by which CBD inhibits the skeletal muscle, Nav1.4. Our results suggest CBD modulates membrane elasticity and directly interacts with Nav1.4 within its pore.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biophysics↗