Experimental and computational biophysics to identify vasodilator drugs targeted at TRPV2 using agonists based on the probenecid scaffold.

TRP channels are important pharmacological targets in physiopathology. TRPV2 plays distinct roles in cardiac and neuromuscular function, immunity, and metabolism, and is associated with pathologies like muscular dystrophy and cancer. However, TRPV2 pharmacology is unspecific and scarce at best. Using in silico similarity-based chemoinformatics we obtained a set of 270 potential hits for TRPV2 categorized into families based on chemical nature and similarity. Docking the compounds on available rat TRPV2 structures allowed the clustering of drug families in specific ligand binding sites. Starting from a probenecid docking pose in the piperlongumine binding site and using a Gaussian accelerated molecular dynamics approach we have assigned a putative probenecid binding site. In parallel, we measured the EC50 of 7 probenecid derivatives on TRPV2 expressed in Pichia pastoris using a novel medium-throughput Ca2+ influx assay in yeast membranes together with an unbiased and unsupervised data analysis method. We found that 4-(piperidine-1-sulfonyl)-benzoic acid had a better EC50 than probenecid, which is one of the most specific TRPV2 agonists to date. Exploring the TRPV2-dependent anti-hypertensive potential in vivo, we found that 4-(piperidine-1-sulfonyl)-benzoic acid shows a sex-biased vasodilator effect producing larger vascular relaxations in female mice. Overall, this study expands the pharmacological toolbox for TRPV2, a widely expressed membrane protein and orphan drug target.

Biallelic variants in SLC4A10 encoding a sodium-dependent bicarbonate transporter lead to a neurodevelopmental disorder.

PURPOSE: SLC4A10 encodes a plasma membrane-bound transporter, which mediates Na+-dependent HCO3- import, thus mediating net acid extrusion. Slc4a10 knockout mice show collapsed brain ventricles, an increased seizure threshold, mild behavioral abnormalities, impaired vision, and deafness. METHODS: Utilizing exome/genome sequencing in families with undiagnosed neurodevelopmental disorders and international data sharing, 11 patients from 6 independent families with biallelic variants in SLC4A10 were identified. Clinico-radiological and dysmorphology assessments were conducted. A minigene assay, localization studies, intracellular pH recordings, and protein modeling were performed to study the possible functional consequences of the variant alleles. RESULTS: The families harbor 8 segregating ultra-rare biallelic SLC4A10 variants (7 missense and 1 splicing). Phenotypically, patients present with global developmental delay/intellectual disability and central hypotonia, accompanied by variable speech delay, microcephaly, cerebellar ataxia, facial dysmorphism, and infrequently, epilepsy. Neuroimaging features range from some non-specific to distinct neuroradiological findings, including slit ventricles and a peculiar form of bilateral curvilinear nodular heterotopia. In silico analyses showed 6 of 7 missense variants affect evolutionarily conserved residues. Functional analyses supported the pathogenicity of 4 of 7 missense variants. CONCLUSION: We provide evidence that pathogenic biallelic SLC4A10 variants can lead to neurodevelopmental disorders characterized by variable abnormalities of the central nervous system, including altered brain ventricles, thus resembling several features observed in knockout mice.

Trapping of a Polyketide Synthase Module after C-C Bond Formation Reveals Transient Acyl Carrier Domain Interactions.

Modular polyketide synthases (PKSs) are giant assembly lines that produce an impressive range of biologically active compounds. However, our understanding of the structural dynamics of these megasynthases, specifically the delivery of acyl carrier protein (ACP)-bound building blocks to the catalytic site of the ketosynthase (KS) domain, remains severely limited. Using a multipronged structural approach, we report details of the inter-domain interactions after C-C bond formation in a chain-branching module of the rhizoxin PKS. Mechanism-based crosslinking of an engineered module was achieved using a synthetic substrate surrogate that serves as a Michael acceptor. The crosslinked protein allowed us to identify an asymmetric state of the dimeric protein complex upon C-C bond formation by cryo-electron microscopy (cryo-EM). The possible existence of two ACP binding sites, one of them a potential "parking position" for substrate loading, was also indicated by AlphaFold2 predictions. NMR spectroscopy showed that a transient complex is formed in solution, independent of the linker domains, and photochemical crosslinking/mass spectrometry of the standalone domains allowed us to pinpoint the interdomain interaction sites. The structural insights into a branching PKS module arrested after C-C bond formation allows a better understanding of domain dynamics and provides valuable information for the rational design of modular assembly lines.

Crosstalk between regulatory elements in disordered TRPV4 N-terminus modulates lipid-dependent channel activity.

Intrinsically disordered regions (IDRs) are essential for membrane receptor regulation but often remain unresolved in structural studies. TRPV4, a member of the TRP vanilloid channel family involved in thermo- and osmosensation, has a large N-terminal IDR of approximately 150 amino acids. With an integrated structural biology approach, we analyze the structural ensemble of the TRPV4 IDR and the network of antagonistic regulatory elements it encodes. These modulate channel activity in a hierarchical lipid-dependent manner through transient long-range interactions. A highly conserved autoinhibitory patch acts as a master regulator by competing with PIP2 binding to attenuate channel activity. Molecular dynamics simulations show that loss of the interaction between the PIP2-binding site and the membrane reduces the force exerted by the IDR on the structured core of TRPV4. This work demonstrates that IDR structural dynamics are coupled to TRPV4 activity and highlights the importance of IDRs for TRP channel function and regulation.

[4.3.1]Bicyclic FKBP Ligands Inhibit Legionella Pneumophila Infection by LpMip-Dependent and LpMip-Independent Mechanisms.

Legionella pneumophila is the causative agent of Legionnaires' disease, a serious form of pneumonia. Its macrophage infectivity potentiator (Mip), a member of a highly conserved family of FK506-binding proteins (FKBPs), plays a major role in the proliferation of the gram-negative bacterium in host organisms. In this work, we test our library of >1000 FKBP-focused ligands for inhibition of LpMip. The [4.3.1]-bicyclic sulfonamide turned out as a highly preferred scaffold and provided the most potent LpMip inhibitors known so far. Selected compounds were non-toxic to human cells, displayed antibacterial activity and block bacterial proliferation in cellular infection-assays as well as infectivity in human lung tissue explants. The results confirm [4.3.1]-bicyclic sulfonamides as anti-legionellal agents, although their anti-infective properties cannot be explained by inhibition of LpMip alone.

The effects of allosteric and competitive inhibitors on ZIKV protease conformational dynamics explored through smFRET, nanoDSF, DSF, and 19F NMR.

Zika and dengue viruses cause mosquito-borne diseases of high epidemic relevance. The viral NS2B-NS3 proteases play crucial roles in the pathogen replication cycle and are validated drug targets. They can adopt at least two conformations depending on the position of the NS2B cofactor. Recently, we reported ligand-induced conformational changes of dengue virus NS2B-NS3 protease by single-molecule Förster resonance energy transfer (smFRET). Here, we investigated the conformational dynamics of the homologous Zika virus protease through an integrated methodological approach combining smFRET, thermal shift assays (DSF and nanoDSF) and 19F NMR spectroscopy. Our results show that allosteric inhibitors favor the open conformation and competitive inhibitors stabilize the closed conformation of the Zika virus protease.

Functional modulation of chemical mediators in microbial communities.

Interactions between microorganisms are often mediated by specialized metabolites. Although the structures and biosynthesis of these compounds may have been elucidated, microbes exist within complex microbiomes and chemical signals can thus also be subject to community-dependent modifications. Increasingly powerful chemical and biological tools allow to shed light on this poorly understood aspect of chemical ecology. We provide an overview of loss-of-function and gain-of-function chemical mediator (CM) modifications within microbial multipartner relationships. Although loss-of-function modifications are abundant in the literature, few gain-of-function modifications have been described despite their important role in microbial interactions. Research in this field holds great potential for our understanding of microbial interactions and may also provide novel tools for targeted interference with microbial signaling.

Cannabinoid non-cannabidiol site modulation of TRPV2 structure and function.

TRPV2 is a ligand-operated temperature sensor with poorly defined pharmacology. Here, we combine calcium imaging and patch-clamp electrophysiology with cryo-electron microscopy (cryo-EM) to explore how TRPV2 activity is modulated by the phytocannabinoid Δ9-tetrahydrocannabiorcol (C16) and by probenecid. C16 and probenecid act in concert to stimulate TRPV2 responses including histamine release from rat and human mast cells. Each ligand causes distinct conformational changes in TRPV2 as revealed by cryo-EM. Although the binding for probenecid remains elusive, C16 associates within the vanilloid pocket. As such, the C16 binding location is distinct from that of cannabidiol, partially overlapping with the binding site of the TRPV2 inhibitor piperlongumine. Taken together, we discover a new cannabinoid binding site in TRPV2 that is under the influence of allosteric control by probenecid. This molecular insight into ligand modulation enhances our understanding of TRPV2 in normal and pathophysiology.

Analysis of hyperforin (St. John's wort) action at TRPC6 channel leads to the development of a new class of antidepressant drugs.

St. John's wort is an herb, long used in folk medicine for the treatment of mild depression. Its antidepressant constituent, hyperforin, has properties such as chemical instability and induction of drug-drug interactions that preclude its use for individual pharmacotherapies. Here we identify the transient receptor potential canonical 6 channel (TRPC6) as a druggable target to control anxious and depressive behavior and as a requirement for hyperforin antidepressant action. We demonstrate that TRPC6 deficiency in mice not only results in anxious and depressive behavior, but also reduces excitability of hippocampal CA1 pyramidal neurons and dentate gyrus granule cells. Using electrophysiology and targeted mutagenesis, we show that hyperforin activates the channel via a specific binding motif at TRPC6. We performed an analysis of hyperforin action to develop a new antidepressant drug that uses the same TRPC6 target mechanism for its antidepressant action. We synthesized the hyperforin analog Hyp13, which shows similar binding to TRPC6 and recapitulates TRPC6-dependent anxiolytic and antidepressant effects in mice. Hyp13 does not activate pregnan-X-receptor (PXR) and thereby loses the potential to induce drug-drug interactions. This may provide a new approach to develop better treatments for depression, since depression remains one of the most treatment-resistant mental disorders, warranting the development of effective drugs based on naturally occurring compounds.

Development of Reduced Peptide Bond Pseudopeptide Michael Acceptors for the Treatment of Human African Trypanosomiasis.

Human African Trypanosomiasis (HAT) is an endemic protozoan disease widespread in the sub-Saharan region that is caused by T. b. gambiense and T. b. rhodesiense. The development of molecules targeting rhodesain, the main cysteine protease of T. b. rhodesiense, has led to a panel of inhibitors endowed with micro/sub-micromolar activity towards the protozoa. However, whilst impressive binding affinity against rhodesain has been observed, the limited selectivity towards the target still remains a hard challenge for the development of antitrypanosomal agents. In this paper, we report the synthesis, biological evaluation, as well as docking studies of a series of reduced peptide bond pseudopeptide Michael acceptors (SPR10-SPR19) as potential anti-HAT agents. The new molecules show Ki values in the low-micro/sub-micromolar range against rhodesain, coupled with k2nd values between 1314 and 6950 M-1 min-1. With a few exceptions, an appreciable selectivity over human cathepsin L was observed. In in vitro assays against T. b. brucei cultures, SPR16 and SPR18 exhibited single-digit micromolar activity against the protozoa, comparable to those reported for very potent rhodesain inhibitors, while no significant cytotoxicity up to 70 µM towards mammalian cells was observed. The discrepancy between rhodesain inhibition and the antitrypanosomal effect could suggest additional mechanisms of action. The biological characterization of peptide inhibitor SPR34 highlights the essential role played by the reduced bond for the antitrypanosomal effect. Overall, this series of molecules could represent the starting point for further investigations of reduced peptide bond-containing analogs as potential anti-HAT agents.

Extent of intrinsic disorder and NMR chemical shift assignments of the distal N-termini from human TRPV1, TRPV2 and TRPV3 ion channels.

The mammalian Transient Receptor Potential Vanilloid (TRPV) channels are a family of six tetrameric ion channels localized at the plasma membrane. The group I members of the family, TRPV1 through TRPV4, are heat-activated and exhibit remarkable polymodality. The distal N-termini of group I TRPV channels contain large intrinsically disordered regions (IDRs), ranging from ~ 75 amino acids (TRPV2) to ~ 150 amino acids (TRPV4), the vast majority of which is invisible in the structural models published so far. These IDRs provide important binding sites for cytosolic partners, and their deletion is detrimental to channel activity and regulation. Recently, we reported the NMR backbone assignments of the distal TRPV4 N-terminus and noticed some discrepancies between the extent of disorder predicted solely based on protein sequence and from experimentally determined chemical shifts. Thus, for an analysis of the extent of disorder in the distal N-termini of all group I TRPV channels, we now report the NMR assignments for the human TRPV1, TRPV2 and TRPV3 IDRs.

New peptidomimetic rhodesain inhibitors with improved selectivity towards human cathepsins.

Parasitic cysteine proteases such as rhodesain (TbCatL) from Trypanosoma brucei rhodesiense are relevant targets for developing new potential drugs against parasitic diseases (e. g. Human African Trypanosomiasis). Designing selective inhibitors for parasitic cathepsins can be challenging as they share high structural similarities with human cathepsins. In this paper, we describe the development of novel peptidomimetic rhodesain inhibitors by applying a structure-based de novo design approach and molecular docking protocols. The inhibitors with a new scaffold in P2 and P3 position display high selectivity towards trypanosomal rhodesain over human cathepsins L and B and high antitrypanosomal activity. Vinylsulfonate 2a has emerged as a potent rhodesain inhibitor (k2nd = 883 • 103 M-1 s-1) with single-digit nanomolar binding affinity (Ki = 9 nM) and more than 150-fold selectivity towards human cathepsins and it thus constitutes an interesting starting compound for the further development of selective drugs against Human African Trypanosomiasis.

Backbone NMR assignments of the extensive human and chicken TRPV4 N-terminal intrinsically disordered regions as important players in ion channel regulation.

Transient receptor potential (TRP) channels are important pharmacological targets due to their ability to act as sensory transducers on the organismic and cellular level, as polymodal signal integrators and because of their role in numerous diseases. However, a detailed molecular understanding of the structural dynamics of TRP channels and their integration into larger cellular signalling networks remains challenging, in part due to the systematic absence of highly dynamic regions pivotal for channel regulation from available structures. In human TRP vanilloid 4 (TRPV4), a ubiquitously expressed homotetrameric cation channel involved in temperature, osmo- and mechano-sensation and in a multitude of (patho)physiological processes, the intrinsically disordered N-terminus encompasses 150 amino acids and thus represents > 17% of the entire channel sequence. Its deletion renders the channel significantly less excitable to agonists supporting a crucial role in TRPV4 activation and regulation. For a structural understanding and a comparison of its properties across species, we determined the NMR backbone assignments of the human and chicken TRPV4 N-terminal IDRs.

Development of novel dipeptide nitriles as inhibitors of rhodesain of Trypanosoma brucei rhodesiense.

In this paper, we developed a new series of dipeptide nitriles that were demonstrated to be reversible rhodesain inhibitors at nanomolar level, with EC50 values against cultured T. b. brucei in the micromolar range. We also proved that our dipeptide nitriles directly bind to the active site of rhodesain acting as competitive inhibitors. Within the most interesting compounds, the dipeptide nitrile 2b showed the highest binding affinity towards rhodesain (Ki = 16 nM) coupled with a good antiparasitic activity (EC50 = 14.1 µM). Moreover, for the dipeptide nitrile 3e, which showed a Ki = 122 nM towards the trypanosomal protease, we obtained the highest antiparasitic activity (EC50 = 8.8 µM). Thus, given the obtained results both compounds could certainly represent new lead compounds for the discovery of new drugs to treat Human African Trypanosomiasis.

Multiubiquitination of TRPV4 reduces channel activity independent of surface localization.

Ubiquitin (Ub)-mediated regulation of plasmalemmal ion channel activity canonically occurs via stimulation of endocytosis. Whether ubiquitination can modulate channel activity by alternative mechanisms remains unknown. Here, we show that the transient receptor potential vanilloid 4 (TRPV4) cation channel is multiubiquitinated within its cytosolic N-terminal and C-terminal intrinsically disordered regions (IDRs). Mutagenizing select lysine residues to block ubiquitination of the N-terminal but not C-terminal IDR resulted in a marked elevation of TRPV4-mediated intracellular calcium influx, without increasing cell surface expression levels. Conversely, enhancing TRPV4 ubiquitination via expression of an E3 Ub ligase reduced TRPV4 channel activity but did not decrease plasma membrane abundance. These results demonstrate Ub-dependent regulation of TRPV4 channel function independent of effects on plasma membrane localization. Consistent with ubiquitination playing a key negative modulatory role of the channel, gain-of-function neuropathy-causing mutations in the TRPV4 gene led to reduced channel ubiquitination in both cellular and Drosophila models of TRPV4 neuropathy, whereas increasing mutant TRPV4 ubiquitination partially suppressed channel overactivity. Together, these data reveal a novel mechanism via which ubiquitination of an intracellular flexible IDR domain modulates ion channel function independently of endocytic trafficking and identify a contributory role for this pathway in the dysregulation of TRPV4 channel activity by neuropathy-causing mutations.

Total Synthesis and Structure Correction of the Cyclic Lipodepsipeptide Orfamide A.

A total synthesis of the cyclic lipodepsipeptide natural product orfamide A was achieved. By developing a synthesis format using an aminoacid ester building block and SPPS protocol adaptation, a focused library of target compounds was obtained, in high yield and purity. Spectral and LC-HRMS data of all library members with the isolated natural product identified the 5 Leu residue to be d- and the 3'-OH group to be R-configured. The structural correction of orfamide A by chemical synthesis and analysis was confirmed by biological activity comparison in Chlamydomonas reinhardtii, which indicated compound configuration to be important for bioactivity. Acute toxicity was also found against Trypanosoma brucei, the parasite causing African sleeping sickness.

TRPV4 mutations causing mixed neuropathy and skeletal phenotypes result in severe gain of function.

OBJECTIVE: Distinct dominant mutations in the calcium-permeable ion channel TRPV4 (transient receptor potential vanilloid 4) typically cause nonoverlapping diseases of either the neuromuscular or skeletal systems. However, accumulating evidence suggests that some patients develop mixed phenotypes that include elements of both neuromuscular and skeletal disease. We sought to define the genetic and clinical features of these patients. METHODS: We report a 2-year-old with a novel R616G mutation in TRPV4 with a severe neuropathy phenotype and bilateral vocal cord paralysis. Interestingly, a different substitution at the same residue, R616Q, has been reported in families with isolated skeletal dysplasia. To gain insight into clinical features and potential genetic determinants of mixed phenotypes, we perform in-depth analysis of previously reported patients along with functional and structural assessment of selected mutations. RESULTS: We describe a wide range of neuromuscular and skeletal manifestations and highlight specific mutations that are more frequently associated with overlap syndromes. We find that mutations causing severe, mixed phenotypes have an earlier age of onset and result in more marked elevations of intracellular calcium, increased cytotoxicity, and reduced sensitivity to TRPV4 antagonism. Structural analysis of the two mutations with the most dramatic gain of ion channel function suggests that these mutants likely cause constitutive channel opening through disruption of the TRPV4 S5 transmembrane domain. INTERPRETATION: These findings demonstrate that the degree of baseline calcium elevation correlates with development of mixed phenotypes and sensitivity to pharmacologic channel inhibition, observations that will be critical for the design of future clinical trials for TRPV4 channelopathies.

Backbone NMR assignment of the nucleotide binding domain of the Bacillus subtilis ABC multidrug transporter BmrA in the post-hydrolysis state.

ATP binding cassette (ABC) proteins are present in all phyla of life and form one of the largest protein families. The Bacillus subtilis ABC transporter BmrA is a functional homodimer that can extrude many different harmful compounds out of the cell. Each BmrA monomer is composed of a transmembrane domain (TMD) and a nucleotide binding domain (NBD). While the TMDs of ABC transporters are sequentially diverse, the highly conserved NBDs harbor distinctive conserved motifs that enable nucleotide binding and hydrolysis, interdomain communication and that mark a protein as a member of the ABC superfamily. In the catalytic cycle of an ABC transporter, the NBDs function as the molecular motor that fuels substrate translocation across the membrane via the TMDs and are thus pivotal for the entire transport process. For a better understanding of the structural and dynamic consequences of nucleotide interactions within the NBD at atomic resolution, we determined the 1H, 13C and 15N backbone chemical shift assignments of the 259 amino acid wildtype BmrA-NBD in its post-hydrolytic, ADP-bound state.

TRP channels: branching out into the fungal kingdom.

TRP channels have been heavily pursued as cryo-electron microscopy targets since they rang in the "resolution revolution." Although widespread in eukaryotes, a fungal TRP channel structure was missing. In this issue of Structure, Ahmed et al. (2022) present structural insights into the regulation of yeast TRPY1 by Ca2+ and lipids.