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Several novel chemical series were identified that modulate glucocerebrosidase (GCase). Compounds from these series are active on glucosylceramide, unlike other known GCase modulators. We obtained GCase crystal structures with two compounds that have distinct chemotypes. Positive allosteric modulators bind to a site on GCase and induce conformational changes, but also induce an equilibrium state between monomer and dimer., (© 2024 Wiley‐VCH GmbH.)

The human genome encodes 850 G protein-coupled receptors (GPCRs), half of which are considered potential drug targets. GPCRs transduce extracellular stimuli into a plethora of vital physiological processes. Consequently, GPCRs are an attractive drug target class. This is underlined by the fact that approximately 40% of marketed drugs modulate GPCRs. Intriguingly 60% of non-olfactory GPCRs have no drugs or candidates in clinical development, highlighting the continued potential of GPCRs as drug targets. The discovery of small molecules targeting these GPCRs by conventional high throughput screening (HTS) campaigns is challenging. Although the definition of success varies per company, the success rate of HTS for GPCRs is low compared to other target families (Fujioka and Omori, 2012; Dragovich et al., 2022). Beyond this, GPCR structure determination can be difficult, which often precludes the application of structure-based drug design approaches to arising HTS hits. GPCR structural studies entail the resource-demanding purification of native receptors, which can be challenging as they are inherently unstable when extracted from the lipid matrix. Moreover, GPCRs are flexible molecules that adopt distinct conformations, some of which need to be stabilized if they are to be structurally resolved. The complexity of targeting distinct therapeutically relevant GPCR conformations during the early discovery stages contributes to the high attrition rates for GPCR drug discovery programs. Multiple strategies have been explored in an attempt to stabilize GPCRs in distinct conformations to better understand their pharmacology. This review will focus on the use of camelid-derived immunoglobulin single variable domains (VHHs) that stabilize disease-relevant pharmacological states (termed ConfoBodies by the authors) of GPCRs, as well as GPCR:signal transducer complexes, to accelerate drug discovery. These VHHs are powerful tools for supporting in vitro screening, deconvolution of complex GPCR pharmacology, and structural biology purposes. In order to demonstrate the potential impact of ConfoBodies on translational research, examples are presented of their role in active state screening campaigns and structure-informed rational design to identify de novo chemical space and, subsequently, how such matter can be elaborated into more potent and selective drug candidates with intended pharmacology., Competing Interests: TL, ZS, PC, ADB, SDC, ST, AB, DF, AK, V-PJ, and CM are employees or subscription right holders of Confo Therapeutics NV. “ConfoBody” and “ConfoBodies” are registered trademarks of Confo Therapeutics NV., (Copyright © 2022 Laeremans, Sands, Claes, De Blieck, De Cesco, Triest, Busch, Felix, Kumar, Jaakola and Menet.)

Dopamine D1 receptor (D1R) is an important drug target implicated in many psychiatric and neurological disorders. Selective agonism of D1R are sought to be the therapeutic strategy for these disorders. Most selective D1R agonists share a dopamine-like catechol moiety in their molecular structure, and their therapeutic potential is therefore limited by poor pharmacological properties in vivo. Recently, a class of non-catechol D1R selective agonists with a distinct scaffold and pharmacological properties were reported. Here, we report the crystal structure of D1R in complex with stimulatory G protein (Gs) and a non-catechol agonist Compound 1 at 3.8 Å resolution. The structure reveals the ligand bound to D1R in an extended conformation, spanning from the orthosteric site to extracellular loop 2 (ECL2). Structural analysis reveals that the unique features of D1R ligand binding pocket explains the remarkable selectivity of this scaffold for D1R over other aminergic receptors, and sheds light on the mechanism for D1R activation by the non-catechol agonist.

Cryptic pockets are protein cavities that remain hidden in resolved apo structures and generally require the presence of a co-crystallized ligand to become visible. Finding new cryptic pockets is crucial for structure-based drug discovery to identify new ways of modulating protein activity and thus expand the druggable space. We present here a new method and associated web application leveraging mixed-solvent molecular dynamics (MD) simulations using benzene as a hydrophobic probe to detect cryptic pockets. Our all-atom MD-based workflow was systematically tested on 18 different systems and 5 additional kinases and represents the largest validation study of this kind. CrypticScout identifies benzene probe binding hotspots on a protein surface by mapping probe occupancy, residence time, and the benzene occupancy reweighed by the residence time. The method is presented to the scientific community in a web application available via www.playmolecule.org using a distributed computing infrastructure to perform the simulations.

G-protein coupled receptors (GPCRs) play a pivotal role in transmitting signals at the cellular level. Structural insights can be exploited to support GPCR structure-based drug discovery endeavours. Despite advances in GPCR crystallography, active state structures are scarce. Molecular dynamics (MD) simulations have been used to explore the conformational landscape of GPCRs. Efforts have been made to retrieve active state conformations starting from inactive structures, however to date this has not been possible without using an energy bias. Here, we reconstruct the activation pathways of the apo adenosine receptor (A2A), starting from an inactive conformation, by applying adaptive sampling MD combined with a goal-oriented scoring function. The reconstructed pathways reconcile well with experiments and help deepen our understanding of A2A regulatory mechanisms. Exploration of the apo conformational landscape of A2A reveals the existence of ligand-competent states, active intermediates and state-dependent cholesterol hotspots of relevance for drug discovery. To the best of our knowledge this is the first time an activation process has been elucidated for a GPCR starting from an inactive structure only, using a non-biased MD approach, opening avenues for the study of ligand binding to elusive yet pharmacologically relevant GPCR states.

The widely expressed G-protein coupled receptors (GPCRs) are versatile signal transducer proteins that are attractive drug targets but structurally challenging to study. GPCRs undergo a number of conformational rearrangements when transitioning from the inactive to the active state but have so far been believed to adopt a fairly conserved inactive conformation. Using 19 F NMR spectroscopy and advanced molecular dynamics simulations we describe a novel inactive state of the adenosine 2A receptor which is stabilised by the aminotriazole antagonist Cmpd-1. We demonstrate that the ligand stabilises a unique conformation of helix V and present data on the putative binding mode of the compound involving contacts to the transmembrane bundle as well as the extracellular loop 2., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Brivaracetam (BRV) and levetiracetam (LEV) are effective antiepileptic drugs that bind selectively to the synaptic vesicle 2A (SV2A) protein. BRV differs from LEV in preclinical studies in that it exhibits a more potent and complete seizure protection across animal models. We reported previously that an allosteric modulator of the SV2A protein had differential effects on BRV compared with LEV, suggesting that they act at different sites or with different conformations of the SV2A protein. If this is the case, then we hypothesized that mutations of specific amino acids in the SV2A protein may have differential effects on BRV and LEV binding by the modulator. Mutation of some amino acids identified previously in the binding site of racetams to the SV2A protein had marked effects on binding of both [ 3 H]BRV and [ 3 H]LEV (eg, W300F, F277A, G303A, F658A, Y462A, W666A, I663A, D670A, and V661A). However, 3 amino acids were identified (K694, I273, and S294) in which mutation lost the effect of the modulator on [ 3 H]LEV binding with no effect on the modulation of [ 3 H]BRV binding. These results confirm that BRV and LEV bind to the human synaptic vesicle 2A protein at closely related sites but interact with these sites in a different way., (Wiley Periodicals, Inc. © 2018 International League Against Epilepsy.)

De novo gain of function mutations in GRIN2B encoding the GluN2B subunit of the N-methyl-d-aspartate (NMDA) receptor have been linked with epileptic encephalopathies, including infantile spasms. We investigated the effects of radiprodil, a selective GluN2B negative allosteric modulator and other non-selective NMDA receptor inhibitors on glutamate currents mediated by NMDA receptors containing mutated GluN2B subunits. The experiments were performed in Xenopus oocytes co-injected with the following human mRNAs: GRIN1/GRIN2B, GRIN1/GRIN2B-R540H, GRIN1/GRIN2B-N615I and GRIN1/GRIN2B-V618G. Glutamate displayed slightly increased potency in the R540H variant, but not in N615I and V618G variants. However, the inhibition by Mg 2+ was completely abolished in N615I and V618G variants. In fact, Mg 2+ enhanced glutamate responses in those variants. The potency of radiprodil to block glutamate-evoked currents was not affected in any of the variants, while the effects by non-selective NMDA inhibitors were greatly reduced in some of the variants. Additionally, in the Mg 2+ insensitive variants, radiprodil blocked glutamate-activated currents with the same potency as in the absence of Mg 2+ . The gain of function observed in the reported GRIN2B variants could be a key pathophysiological factor leading to neuronal hyper-excitability in epileptic encephalopathies. The GluN2B-selective inhibitor radiprodil fully retained its pharmacological profile under these conditions, while other non-selective NMDA receptor antagonists lost their potency. Consequently, our data suggest that radiprodil may be a valuable therapeutic option for treatment of pediatric epileptic encephalopathies associated with GRIN2B mutations., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The adenosine A 2A receptor (A 2A R) has long been implicated in cardiovascular disorders. As more selective A 2A R ligands are being identified, its roles in other disorders, such as Parkinson's disease, are starting to emerge, and A 2A R antagonists are important drug candidates for nondopaminergic anti-Parkinson treatment. Here we report the crystal structure of A 2A receptor bound to compound 1 (Cmpd-1), a novel A 2A R/ N -methyl d-aspartate receptor subtype 2B (NR2B) dual antagonist and potential anti-Parkinson candidate compound, at 3.5 Å resolution. The A 2A receptor with a cytochrome b562-RIL (BRIL) fusion (A 2A R-BRIL) in the intracellular loop 3 (ICL3) was crystallized in detergent micelles using vapor-phase diffusion. Whereas A 2A R-BRIL bound to the antagonist ZM241385 has previously been crystallized in lipidic cubic phase (LCP), structural differences in the Cmpd-1-bound A 2A R-BRIL prevented formation of the lattice observed with the ZM241385-bound receptor. The crystals grew with a type II crystal lattice in contrast to the typical type I packing seen from membrane protein structures crystallized in LCP. Cmpd-1 binds in a position that overlaps with the native ligand adenosine, but its methoxyphenyl group extends to an exosite not previously observed in other A 2A R structures. Structural analysis revealed that Cmpd-1 binding results in the unique conformations of two tyrosine residues, Tyr9 1.35 and Tyr271 7.36 , which are critical for the formation of the exosite. The structure reveals insights into antagonist binding that are not observed in other A 2A R structures, highlighting flexibility in the binding pocket that may facilitate the development of A 2A R-selective compounds for the treatment of Parkinson's disease.

The synaptic vesicle glycoprotein SV2A belongs to the major facilitator superfamily (MFS) of transporters and is an integral constituent of synaptic vesicle membranes. SV2A has been demonstrated to be involved in vesicle trafficking and exocytosis, processes crucial for neurotransmission. The anti-seizure drug levetiracetam was the first ligand to target SV2A and displays a broad spectrum of anti-seizure activity in various preclinical models. Several lines of preclinical and clinical evidence, including genetics and protein expression changes, support an important role of SV2A in epilepsy pathophysiology. While the functional consequences of SV2A ligand binding are not fully elucidated, studies suggest that subsequent SV2A conformational changes may contribute to seizure protection. Conversely, the recently discovered negative SV2A modulators, such as UCB0255, counteract the anti-seizure effect of levetiracetam and display procognitive properties in preclinical models. More broadly, dysfunction of SV2A may also be involved in Alzheimer's disease and other types of cognitive impairment, suggesting potential novel therapies for levetiracetam and its congeners. Furthermore, emerging data indicate that there may be important roles for two other SV2 isoforms (SV2B and SV2C) in the pathogenesis of epilepsy, as well as other neurodegenerative diseases. Utilization of recently developed SV2A positron emission tomography ligands will strengthen and reinforce the pharmacological evidence that SV2A is a druggable target, and will provide a better understanding of its role in epilepsy and other neurological diseases, aiding in further defining the full therapeutic potential of SV2A modulation., Competing Interests: Compliance with Ethical Standards Funding Wolfgang Löscher’s initial animal studies on levetiracetam were supported by grants from UCB Pharma. No funding was obtained for preparing this current review. The open access payment for this article was funded by UCB Pharma. Conflict of interest Michel Gillard, Zara Sands, Rafal M. Kaminski, and Henrik Klitgaard are employees of UCB Pharma (Braine-l’Alleud, Belgium). Wolfgang Löscher has no conflicts of interest to declare.

The Major Facilitator Superfamily (MFS) is one of the largest classes of secondary active transporters and is widely expressed in many domains of life. It is characterized by a common 12-transmembrane helix motif that allows the selective transport of a vast range of diverse substrates across the membrane. MFS transporters play a central role in many physiological processes and are increasingly recognized as potential drug targets. Despite intensive efforts, there are still only a handful of crystal structures and therefore homology modeling is likely to be a necessary process for providing models to interpret experiments for many years to come. However, the diversity of sequences and the multiple conformational states these proteins can exist in makes the process significantly more complicated, especially for sequences for which there is very little sequence identity to known templates. Inspired by the approach adopted many years ago for GPCRs, we have analyzed the large number of MFS sequences now available alongside the current structural information to propose a series of conserved contact points that can provide additional guidance for the homology modeling process. To enable cross-comparison across MFS models we also present a numbering scheme that can be used to provide a point of reference within each of the 12 transmembrane regions.

A necessary step prior to starting any membrane protein computer simulation is the creation of a well-packed configuration of protein(s) and lipids. Here, we demonstrate a method, alchembed , that can simultaneously and rapidly embed multiple proteins into arrangements of lipids described using either atomistic or coarse-grained force fields. During a short simulation, the interactions between the protein(s) and lipids are gradually switched on using a soft-core van der Waals potential. We validate the method on a range of membrane proteins and determine the optimal soft-core parameters required to insert membrane proteins. Since all of the major biomolecular codes include soft-core van der Waals potentials, no additional code is required to apply this method. A tutorial is included in the Supporting Information.

The putative Major Facilitator Superfamily (MFS) transporter, SV2A, is the target for levetiracetam (LEV), which is a successful anti-epileptic drug. Furthermore, SV2A knock out mice display a severe seizure phenotype and die after a few weeks. Despite this, the mode of action of LEV is not known at the molecular level. It would be extremely desirable to understand this more fully in order to aid the design of improved anti-epileptic compounds. Since there is no structure for SV2A, homology modelling can provide insight into the ligand-binding site. However, it is not a trivial process to build such models, since SV2A has low sequence identity to those MFS transporters whose structures are known. A further level of complexity is added by the fact that it is not known which conformational state of the receptor LEV binds to, as multiple conformational states have been inferred by tomography and ligand binding assays or indeed, if binding is exclusive to a single state. Here, we explore models of both the inward and outward facing conformational states of SV2A (according to the alternating access mechanism for MFS transporters). We use a sequence conservation analysis to help guide the homology modelling process and generate the models, which we assess further with Molecular Dynamics (MD). By comparing the MD results in conjunction with docking and simulation of a LEV-analogue used in radioligand binding assays, we were able to suggest further residues that line the binding pocket. These were confirmed experimentally. In particular, mutation of D670 leads to a complete loss of binding. The results shed light on the way LEV analogues may interact with SV2A and may help with the on-going design of improved anti-epileptic compounds.

An X-ray crystal structure of Kelch-like ECH-associated protein (Keap1) co-crystallised with (1S,2R)-2-[(1S)-1-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1,2,3,4-tetrahydroisoquinolin-2-carbonyl]cyclohexane-1-carboxylic acid (compound (S,R,S)-1 a) was obtained. This X-ray crystal structure provides breakthrough experimental evidence for the true binding mode of the hit compound (S,R,S)-1 a, as the ligand orientation was found to differ from that of the initial docking model, which was available at the start of the project. Crystallographic elucidation of this binding mode helped to focus and drive the drug design process more effectively and efficiently., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Congenital Insensitivity to Pain (CIP) is a loss of function mutation resulting in a truncated NaV1.7 protein, suggesting a pivotal role in pain signaling and rendering it an important pharmaceutical target for multiple pain conditions. The structural homology in the NaV-channel family makes it challenging to design effective analgesic compounds without inducing for example cardiotoxicity or seizure liabilities. An additional approach to structural isoform selectivity is to identify compounds with use- or state-dependent profiles, i.e. inhibition efficacy based on the gating of the ion channel. In general nerve cells in damaged or inflamed tissue are more depolarized and electrically active compared to healthy nerve cells in for instance the heart. This observation has led to the design of two types of screening protocols emulating the voltage condition of peripheral neurons or cardiac tissue. The two voltage protocols have been developed to identify both use- and state-dependent antagonists. In this paper we describe an attempt to merge the two different protocols into one to increase screening efficacy, while retaining relevant state- and use-dependent pharmacology. The new protocol is constructed of two stimulation pulses and a slow voltage ramp for simultaneous assessment of resting and state-dependent block. By comparing all protocols we show that the new protocol indeed filter compounds for state-dependence and increase the prediction power of selecting use-dependent compounds.

Hv1 voltage-gated proton channels mediate rapid and selective transmembrane H(+) flux and are gated by both voltage and pH gradients. Selective H(+) transfer in membrane proteins is commonly achieved by Grotthuss proton 'hopping' in chains of ionizable amino acid side chains and intraprotein water molecules. To identify whether ionizable residues are required for proton permeation in Hv1, we neutralized candidate residues and measured expressed voltage-gated H(+) currents. Unexpectedly, charge neutralization was insufficient to abrogate either the Hv1 conductance or coupling of pH gradient and voltage-dependent activation. Molecular dynamics simulations revealed water molecules in the central crevice of Hv1 model structures but not in homologous voltage-sensor domain (VSD) structures. Our results indicate that Hv1 most likely forms an internal water wire for selective proton transfer and that interactions between water molecules and S4 arginines may underlie coupling between voltage- and pH-gradient sensing.

Potassium (K (+)) channels can regulate ionic conduction through their pore by a mechanism, involving the selectivity filter, known as C-type inactivation. This process is rapid in the hERG K (+) channel and is fundamental to its physiological role. Although mutations within hERG are known to remove this process, a structural basis for the inactivation mechanism has yet to be characterized. Using MD simulations based on homology modeling, we observe that the carbonyl of the filter aromatic, Phe627, forming the S 0 K (+) binding site, swiftly rotates away from the conduction axis in the wild-type channel. In contrast, in well-characterized non-inactivating mutant channels, this conformational change occurs less frequently. In the non-inactivating channels, interactions with a water molecule located behind the selectivity filter are critical to the enhanced stability of the conducting state. We observe comparable conformational changes in the acid sensitive TASK-1 channel and propose a common mechanism in these channels for regulating efflux of K (+) ions through the selectivity filter.

The nature of voltage sensing by voltage-activated ion channels is a key problem in membrane protein structural biology. The way in which the voltage-sensor (VS) domain interacts with its membrane environment remains unclear. In particular, the known structures of Kv channels do not readily explain how a positively charged S4 helix is able to stably span a lipid bilayer. Extended (2 x 50 ns) molecular dynamics simulations of the high-resolution structure of the isolated VS domain from the archaebacterial potassium channel KvAP, embedded in zwitterionic and in anionic lipid bilayers, have been used to explore VS/lipid interactions at atomic resolution. The simulations reveal penetration of water into the center of the VS and bilayer. Furthermore, there is significant local deformation of the lipid bilayer by interactions between lipid phosphate groups and arginine side chains of S4. As a consequence of this, the electrostatic field is "focused" across the center of the bilayer.

VSTx1 is a tarantula venom toxin which binds to the archaebacterial voltage-gated potassium channel KvAP. VSTx1 is thought to access the voltage sensor domain of the channel via the lipid bilayer phase. In order to understand its mode of action and implications for the mechanism of channel activation, it is important to characterize the interactions of VSTx1 with lipid bilayers. Molecular dynamics (MD) simulations (for a total simulation time in excess of 0.2 micros) have been used to explore VSTx1 localization and interactions with zwitterionic (POPC) and with anionic (POPE/POPG) lipid bilayers. In particular, three series of MD simulations have been used to explore the net drift of VSTx1 relative to the center of a bilayer, starting from different locations of the toxin. The preferred location of the toxin is at the membrane/water interface. Although there are differences between POPC and POPE/POPG bilayers, in both cases the toxin forms favorable interactions at the interface, maximizing H-bonding to lipid headgroups and to water molecules while retaining interactions with the hydrophobic core of the bilayer. A 30 ns unrestrained simulation reveals dynamic partitioning of VSTx1 into the interface of a POPC bilayer. The preferential location of VSTx1 at the interface is discussed in the context of Kv channel gating models and provides support for a mode of action in which the toxin interacts with the Kv voltage sensor "paddle" formed by the S3 and S4 helices.

A novel series of methyl ester-terminated C8-linked pyrrolobenzodiazepine (PBD)-poly(N-methylpyrrole) conjugates (50a-f) has been synthesized and their DNA interaction evaluated by thermal denaturation, DNA footprinting, and in vitro transcription stop assays. The synergistic effect of attaching a PBD unit to a polypyrrole fragment is illustrated by the large increase in DNA binding affinity (up to 50-fold) compared to the individual PBD and pyrrole components. 50a-f were found to bind mainly to identical DNA sequences but with apparent binding site widths increasing with molecular length and the majority of sites conforming to the consensus motif 5'-XGXWz (z = 3 +/- 1; W = A or T; X = any base but preferably a purine). They also provided robust sequence-selective blockade of transcription at sites corresponding approximately to their DNA footprints. 50a-f were shown to have good cellular/nuclear penetration properties, and a degree of correlation between cytotoxicity and DNA-binding affinity was observed.

Analysis of the crystal structures of the intact voltage-sensitive potassium channel KvAP (from Aeropyrum pernix) and Kv1.2 (from rat brain), along with the isolated voltage sensor (VS) domain from KvAP, raises the question of the exact nature of the voltage-sensing conformational change that triggers activation of Kv and related voltage-gated channels. Molecular dynamics simulations of the isolated VS of KvAP in a detergent micelle environment at two different temperatures (300 K and 368 K) have been used to probe the intrinsic flexibility of this domain on a tens-of-nanoseconds timescale. The VS contains a positively charged (S4) helix which is packed against a more hydrophobic S3 helix. The simulations at elevated temperature reveal an intrinsic flexibility/conformational instability of the S3a region (i.e., the C-terminus of the S3 helix). It is also evident that the S4 helix undergoes hinge bending and swiveling about its central I130 residue. The conformational instability of the S3a region facilitates the motion of the N-terminal segment of S4 (i.e., S4a). These simulations thus support a gating model in which, in response to depolarization, an S3b-S4a "paddle" may move relative to the rest of the VS domain. The flexible S3a region may in turn act to help restore the paddle to its initial conformation upon repolarization.

Molecular dynamics simulations may be used to probe the interactions of membrane proteins with lipids and with detergents at atomic resolution. Examples of such simulations for ion channels and for bacterial outer membrane proteins are described. Comparison of simulations of KcsA (an alpha-helical bundle) and OmpA (a beta-barrel) reveals the importance of two classes of side chains in stabilizing interactions with the head groups of lipid molecules: (i) tryptophan and tyrosine; and (ii) arginine and lysine. Arginine residues interacting with lipid phosphate groups play an important role in stabilizing the voltage-sensor domain of the KvAP channel within a bilayer. Simulations of the bacterial potassium channel KcsA reveal specific interactions of phosphatidylglycerol with an acidic lipid-binding site at the interface between adjacent protein monomers. A combination of molecular modelling and simulation reveals a potential phosphatidylinositol 4,5-bisphosphate-binding site on the surface of Kir6.2.

The recently determined structure of a mammalian voltage-gated potassium channel has important implications for our understanding of voltage-sensing and gating mechanisms in channels. It is also the first crystal structure of an overexpressed eukaryotic membrane protein.

Advances in nanomanipulation techniques have made it possible to measure the response of an individual biomolecule to a force applied in the laboratory. Experiments that stretch a single molecule of duplex DNA have been difficult to interpret theoretically, particularly as the major changes in molecular structure caused by the force cannot be measured. In principle, computer simulation can calculate these conformational changes in atomic level detail, but to date such studies have failed to reproduce the experimental data due to the computational expense of the calculations. Here we show that a combination of molecular modeling and statistical physics can be used successfully to understand the stretching behavior of DNA. Our simulations provide new information about the dynamics of DNA denaturation under force in atomic level detail and also show the importance of entropy in determining biomechanical properties in general.