Developing insulin-like peptide 5-based antagonists for the G protein-coupled receptor, RXFP4.

Human insulin-like peptide 5 (INSL5) is a gut hormone produced by colonic L-cells, and its biological functions are mediated by Relaxin Family Peptide Receptor 4 (RXFP4). Our preliminary data indicated that RXFP4 agonists are potential drug leads for the treatment of constipation. More recently, we designed and developed a novel RXFP4 antagonist, A13-nR that was shown to block agonist-induced activity in cells and animal models. We showed that A13-nR was able to block agonist-induced increases in colon motility in mice of both genders that express the receptor, RXFP4. Our data also showed that colorectal propulsion induced by intracolonic administration of short-chain fatty acids was antagonized by A13-nR. Therefore, A13-nR is an important research tool and potential drug lead for the treatment of colon motility disorders, such as bacterial diarrhea. However, A13-nR acted as a partial agonist at high concentrations in vitro and demonstrated modest antagonist potency (∼35 nM). Consequently, the primary objective of this study is to pinpoint novel modifications to A13-nR that eliminate partial agonist effects while preserving or augmenting antagonist potency. In this work, we detail the creation of a series of A13-nR-modified analogues, among which analogues 3, 4, and 6 demonstrated significantly improved RXFP4 affinity (∼3 nM) with reduced partial agonist activity, enhanced antagonist potency (∼10 nM) and maximum agonist inhibition (∼80 %) when compared with A13-nR. These compounds have potential as candidates for further preclinical evaluations, marking a significant stride toward innovative therapeutics for colon motility disorders.

The novel rapid formulation of intravenous dantrolene (NPJ5008) versus standard dantrolene (Dantrium®): A clinical part-randomised phase 1 study in healthy volunteers.

BACKGROUND: Delays in treating anaesthesia-induced malignant hyperthermia increase risks of complications and death. NPJ5008 is a novel formulation of the indicated treatment, dantrolene sodium, developed to shorten preparation and administration times compared with the reference formulation Dantrium®. The two formulations have been compared preclinically. OBJECTIVES: Assess bioequivalence of overall dantrolene (free acid) exposure of NPJ5008 versus Dantrium® and ascertain similarities in their pharmacokinetics and safety/tolerability profiles. Evaluate preparation/administration time savings for the new formulation. DESIGN: Part 1 of this open-label trial in humans was a 1 : 1 randomised crossover study; part 2 was a single-arm study. Trial pharmacy data and laboratory simulations assessed preparation/administration step timings. SETTING: Single clinical centre in the UK, April to July 2021. PARTICIPANTS: Twenty-one healthy male and female individuals. INTERVENTIONS: Part 1: single intravenous 60 mg dose of NPJ5008 or Dantrium®, sequentially. Part 2: single intravenous 120 mg dose of NPJ5008. Simulation: five vials per formulation using paediatric and adult cannulas. MAIN OUTCOME MEASURES: Overall drug exposure to last measurable concentration (AUC 0 to last ) and extrapolated to infinity (AUC 0 to ∞ ) were primary endpoints. Other pharmacokinetic, clinical and muscle-function parameters, and adverse events, were monitored. RESULTS: Adjusted geometric mean ratios of NPJ5008 versus Dantrium® were 90.24 and 90.44% for AUC 0 to last and AUC 0 to ∞ , respectively, with the 90% confidence intervals (CI) within the 80 to 125% acceptance interval, establishing bioequivalence. No new safety issues emerged: any adverse events were of a similar magnitude across treatments and related to pharmacological properties of dantrolene. Pharmacy and simulation data revealed that every step in preparation and administration was 26 to 69% faster for NPJ5008 than Dantrium®. CONCLUSION: NPJ5008 showed comparable pharmacokinetic and safety profiles to Dantrium®, while reducing dantrolene dose preparation/administration times, potentially reducing patient complications/healthcare resourcing in malignant hyperthermia. TRIAL REGISTRATION: EudraCT Number: 2020-005719-35, MHRA approval.

The 8-hydroxyquinoline derivative, clioquinol, is an alpha-1 adrenoceptor antagonist.

Clioquinol (5-chloro-7-iodo-8-hydroxyquinoline) is an antimicrobial agent whose actions as a zinc or copper ionophore and an iron chelator revived the interest in similar compounds for the treatment of fungal and bacterial infections, neurodegeneration and cancer. Recently, we reported zinc ionophores, including clioquinol, cause vasorelaxation in isolated arteries through mechanisms that involve sensory nerves, endothelium and vascular smooth muscle. Here, we report that clioquinol also uniquely acts as a competitive alpha-1 (α1) adrenoceptor antagonist. We employed ex vivo functional vascular contraction and pharmacological techniques in rat isolated mesenteric arteries, receptor binding assays using stabilized solubilized α1 receptor variants, or wild-type human α1-adrenoceptors transfected in COS-7 cells (African green monkey kidney fibroblast-like cells), and molecular dynamics homology modelling based on the recently published α1A adrenoceptor cryo-EM and α1B crystal structures. At higher concentrations, all ionophores including clioquinol cause a non-competitive antagonism of agonist-mediated contraction due to intracellular zinc delivery, as reported previously. However, at lower concentration ranges, clioquinol has an additional mechanism of competitively inhibiting α1-adrenoceptors that contributes to decreasing vascular contractility. Molecular dynamic simulation showed that clioquinol binds stably to the orthosteric binding site (Asp106) of the receptor, confirming the structural basis for competitive α1-adrenoceptor antagonism by clioquinol.

Identification of a Novel Subtype-Selective α1B-Adrenoceptor Antagonist.

α1A-, α1B-, and α1D-adrenoceptors (α1-ARs) are members of the adrenoceptor G protein-coupled receptor family that are activated by adrenaline (epinephrine) and noradrenaline. α1-ARs are clinically targeted using antagonists that have minimal subtype selectivity, such as prazosin and tamsulosin, to treat hypertension and benign prostatic hyperplasia, respectively. Abundant expression of α1-ARs in the heart and central nervous system (CNS) makes these receptors potential targets for the treatment of cardiovascular and CNS disorders, such as heart failure, epilepsy, and Alzheimer's disease. Our understanding of the precise physiological roles of α1-ARs, however, and their involvement in disease has been hindered by the lack of sufficiently subtype-selective tool compounds, especially for α1B-AR. Here, we report the discovery of 4-[(2-hydroxyethyl)amino]-6-methyl-2H-chromen-2-one (Cpd1), as an α1B-AR antagonist that has 10-15-fold selectivity over α1A-AR and α1D-AR. Through computational and site-directed mutagenesis studies, we have identified the binding site of Cpd1 in α1B-AR and propose the molecular basis of α1B-AR selectivity, where the nonconserved V19745.52 residue plays a major role, with contributions from L3146.55 within the α1B-AR pocket. By exploring the structure-activity relationships of Cpd1 at α1B-AR, we have also identified 3-[(cyclohexylamino)methyl]-6-methylquinolin-2(1H)-one (Cpd24), which has a stronger binding affinity than Cpd1, albeit with reduced selectivity for α1B-AR. Cpd1 and Cpd24 represent potential leads for α1B-AR-selective drug discovery and novel tool molecules to further study the physiology of α1-ARs.

David J. Chalmers.

David Chalmers is a philosopher who studies consciousness. After sketching his background in mathematics, science, and philosophy, he describes the problems of consciousness and his collaboration with neuroscientists. He also discusses the roles of neuroscience and philosophy in studying consciousness and other topics as well as the future of these fields.

The computational and the representational language-of-thought hypotheses.

There are two versions of the language-of-thought hypothesis (LOT): Representational LOT (roughly, structured representation), introduced by Ockham, and computational LOT (roughly, symbolic computation) introduced by Fodor. Like many others, I oppose the latter but not the former. Quilty-Dunn et al. defend representational LOT, but they do not defend the strong computational LOT thesis central to the classical-connectionist debate.

Interaction of the Inhibitory Peptides ShK and HmK with the Voltage-Gated Potassium Channel KV1.3: Role of Conformational Dynamics.

Peptide toxins that adopt the ShK fold can inhibit the voltage-gated potassium channel KV1.3 with IC50 values in the pM range and are therefore potential leads for drugs targeting autoimmune and neuroinflammatory diseases. Nuclear magnetic resonance (NMR) relaxation measurements and pressure-dependent NMR have shown that, despite being cross-linked by disulfide bonds, ShK itself is flexible in solution. This flexibility affects the local structure around the pharmacophore for the KV1.3 channel blockade and, in particular, the relative orientation of the key Lys and Tyr side chains (Lys22 and Tyr23 in ShK) and has implications for the design of KV1.3 inhibitors. In this study, we have performed molecular dynamics (MD) simulations on ShK and a close homologue, HmK, to probe the conformational space occupied by the Lys and Tyr residues, and docked the different conformations with a recently determined cryo-EM structure of the KV1.3 channel. Although ShK and HmK have 60% sequence identity, their dynamic behaviors are quite different, with ShK sampling a broad range of conformations over the course of a 5 µs MD simulation, while HmK is relatively rigid. We also investigated the importance of conformational dynamics, in particular the distance between the side chains of the key dyad Lys22 and Tyr23, for binding to KV1.3. Although these peptides have quite different dynamics, the dyad in both adopts a similar configuration upon binding, revealing a conformational selection upon binding to KV1.3 in the case of ShK. Both peptides bind to KV1.3 with Lys22 occupying the pore of the channel. Intriguingly, the more flexible peptide, ShK, binds with significantly higher affinity than HmK.

Molecular Dynamics Simulation Studies of Bile, Bile Salts, Lipid-Based Drug Formulations, and mRNA-Lipid Nanoparticles: A Review.

Lipid-based formulation (LBF) is an effective approach for delivering hydrophobic drugs into the systemic circulation by oral administration. However, much of the physical detail regarding the colloidal behavior of LBFs and their interactions with the contents of the gastrointestinal (GI) environment is not well characterized. Recently, researchers have started to use molecular dynamics (MD) simulations to investigate the colloidal behavior of LBF systems and their interactions with bile and other materials present in the GI tract. MD is a computational method, based on classical mechanics, that simulates the physical movements of atoms and provides atomic-scale information that cannot easily be retrieved using experimental investigations. MD can provide insight into assist the development of drug formulations in a cost and time-effective manner. This review summarizes the application of MD simulation to the study of bile, bile salts, and LBFs and their behavior within the GI environment and also discusses MD simulations of lipid-based mRNA vaccine formulations.

Targeting HIV-1 Reverse Transcriptase Using a Fragment-Based Approach.

Human immunodeficiency virus type I (HIV-1) is a retrovirus that infects cells of the host's immune system leading to acquired immunodeficiency syndrome and potentially death. Although treatments are available to prevent its progression, HIV-1 remains a major burden on health resources worldwide. Continued emergence of drug-resistance mutations drives the need for novel drugs that can inhibit HIV-1 replication through new pathways. The viral protein reverse transcriptase (RT) plays a fundamental role in the HIV-1 replication cycle, and multiple approved medications target this enzyme. In this study, fragment-based drug discovery was used to optimize a previously identified hit fragment (compound B-1), which bound RT at a novel site. Three series of compounds were synthesized and evaluated for their HIV-1 RT binding and inhibition. These series were designed to investigate different vectors around the initial hit in an attempt to improve inhibitory activity against RT. Our results show that the 4-position of the core scaffold is important for binding of the fragment to RT, and a lead compound with a cyclopropyl substitution was selected and further investigated. Requirements for binding to the NNRTI-binding pocket (NNIBP) and a novel adjacent site were investigated, with lead compound 27-a minimal but efficient NNRTI-offering a starting site for the development of novel dual NNIBP-Adjacent site inhibitors.

An adversarial collaboration protocol for testing contrasting predictions of global neuronal workspace and integrated information theory.

The relationship between conscious experience and brain activity has intrigued scientists and philosophers for centuries. In the last decades, several theories have suggested different accounts for these relationships. These theories have developed in parallel, with little to no cross-talk among them. To advance research on consciousness, we established an adversarial collaboration between proponents of two of the major theories in the field, Global Neuronal Workspace and Integrated Information Theory. Together, we devised and preregistered two experiments that test contrasting predictions of these theories concerning the location and timing of correlates of visual consciousness, which have been endorsed by the theories' proponents. Predicted outcomes should either support, refute, or challenge these theories. Six theory-impartial laboratories will follow the study protocol specified here, using three complementary methods: Functional Magnetic Resonance Imaging (fMRI), Magneto-Electroencephalography (M-EEG), and intracranial electroencephalography (iEEG). The study protocol will include built-in replications, both between labs and within datasets. Through this ambitious undertaking, we hope to provide decisive evidence in favor or against the two theories and clarify the footprints of conscious visual perception in the human brain, while also providing an innovative model of large-scale, collaborative, and open science practice.

Targeting Melanocortin Receptors Using SNAr-Type Macrocyclization: A Doubly Orthogonal Route to Cyclic Peptide Conjugates.

While a range of strategies exist to accomplish peptide macrocyclization, they are frequently limited by the need for orthogonal protection or provide little opportunity for structural diversification. We have evaluated an efficient macrocyclization method that employs nucleophilic aromatic substitution (SNAr) to create thioether macrocycles. This versatile macrocyclization, orthogonal to conventional peptide synthesis, can be performed in solution on unprotected peptidomimetics or on resin-bound peptides with side-chain protection in place. We show that the electron-withdrawing groups present in the products can be further utilized in subsequent orthogonal reactions to alter the peptide properties or to add prosthetic groups. The macrocyclization strategy was applied to the design of melanocortin ligands, generating a library of potent melanocortin agonists that exhibit distinct subtype selectivity.

Structural Analysis of the Complex of Human Transthyretin with 3',5'-Dichlorophenylanthranilic Acid at 1.5 Å Resolution.

Human transthyretin (hTTR) can form amyloid deposits that accumulate in nerves and organs, disrupting cellular function. Molecules such as tafamidis that bind to and stabilize the TTR tetramer can reduce such amyloid formation. Here, we studied the interaction of VCP-6 (2-((3,5-dichlorophenyl)amino)benzoic acid) with hTTR. VCP-6 binds to hTTR with 5 times the affinity of the cognate ligand, thyroxine (T4). The structure of the hTTR:VCP-6 complex was determined by X-ray crystallography at 1.52 Šresolution. VCP-6 binds deeper in the binding channel than T4 with the 3',5'-dichlorophenyl ring binding in the 'forward' mode towards the channel centre. The dichlorophenyl ring lies along the 2-fold axis coincident with the channel centre, while the 2-carboxylatephenylamine ring of VCP-6 is symmetrically displaced from the 2-fold axis, allowing the 2-carboxylate group to form a tight intermolecular hydrogen bond with Nζ of Lys15 and an intramolecular hydrogen bond with the amine of VCP-6, stabilizing its conformation and explaining the greater affinity of VCP-6 compared to T4. This arrangement maintains optimal halogen bonding interactions in the binding sites, via chlorine atoms rather than iodine of the thyroid hormone, thereby explaining why the dichloro substitution pattern is a stronger binder than either the diiodo or dibromo analogues.

Membrane Permeating Macrocycles: Design Guidelines from Machine Learning.

The ability to predict cell-permeable candidate molecules has great potential to assist drug discovery projects. Large molecules that lie beyond the Rule of Five (bRo5) are increasingly important as drug candidates and tool molecules for chemical biology. However, such large molecules usually do not cross cell membranes and cannot access intracellular targets or be developed as orally bioavailable drugs. Here, we describe a random forest (RF) machine learning model for the prediction of passive membrane permeation rates developed using a set of over 1000 bRo5 macrocyclic compounds. The model is based on easily calculated chemical features/descriptors as independent variables. Our random forest (RF) model substantially outperforms a multiple linear regression model based on the same features and achieves better performance metrics than previously reported models using the same underlying data. These features include: (1) polar surface area in water, (2) the octanol-water partitioning coefficient, (3) the number of hydrogen-bond donors, (4) the sum of the topological distances between nitrogen atoms, (5) the sum of the topological distances between nitrogen and oxygen atoms, and (6) the multiple molecular path count of order 2. The last three features represent molecular flexibility, the ability of the molecule to adopt different conformations in the aqueous and membrane interior phases, and the molecular "chameleonicity." Guided by the model, we propose design guidelines for membrane-permeating macrocycles. It is anticipated that this model will be useful in guiding the design of large, bioactive molecules for medicinal chemistry and chemical biology applications.

Enhanced nitric oxide production by macrophages treated with a cell-penetrating peptide conjugate.

The SPRY domain-containing SOCS box protein-2 (SPSB2) plays a critical role in the degradation of inducible nitric oxide synthase (iNOS) in macrophages. In this study, we have conjugated a peptide inhibitor of the iNOS-SPSB2 interaction with a cell-penetrating peptide (CPP) for delivery into macrophages, and confirmed its binding to SPSB2. We have assessed the uptake of a fluorophore-tagged analogue by RAW 264.7 and immortalised bone marrow derived macrophage (iBMDM) cell lines, and shown that the CPP-peptide conjugate enhanced NO production. The findings of this study will be useful in further refinement of CPP-peptide conjugates as leads in the development of new antibiotics that target the host innate immune response.

Crystal structure of the α1B-adrenergic receptor reveals molecular determinants of selective ligand recognition.

α-adrenergic receptors (αARs) are G protein-coupled receptors that regulate vital functions of the cardiovascular and nervous systems. The therapeutic potential of αARs, however, is largely unexploited and hampered by the scarcity of subtype-selective ligands. Moreover, several aminergic drugs either show off-target binding to αARs or fail to interact with the desired subtype. Here, we report the crystal structure of human α1BAR bound to the inverse agonist (+)-cyclazosin, enabled by the fusion to a DARPin crystallization chaperone. The α1BAR structure allows the identification of two unique secondary binding pockets. By structural comparison of α1BAR with α2ARs, and by constructing α1BAR-α2CAR chimeras, we identify residues 3.29 and 6.55 as key determinants of ligand selectivity. Our findings provide a basis for discovery of α1BAR-selective ligands and may guide the optimization of aminergic drugs to prevent off-target binding to αARs, or to elicit a selective interaction with the desired subtype.

Resolution of post-circumcision penile adhesions in newborns.

INTRODUCTION: Penile adhesions are a common complication of neonatal circumcision and can cause significant distress to families. Although prior studies have suggested adhesions resolve with conservative measures, the information on the likelihood and time frame is not known. Alternatively, a lysis of adhesions (LOA) procedure can be performed in the office. The aim of the study was to determine the likelihood of spontaneous resolution with conservative measures at home. METHODS: A retrospective review of patients with post circumcision penile adhesions that were referred to a pediatric urologist was conducted. Data regarding patient age, extent and severity of adhesions, chosen treatment modality (home-care with daily retraction versus LOA), improvement in subsequent visits, and final outcome of adhesions was obtained from chart review. Contingency tables, chi-square tests, or t-tests were used to assess statistical significance. RESULTS: 429 patients were reviewed. The mean age was 28.2 months (range 1-152) and mean follow-up was 7.4 months (range 1-70). Adhesions were circumferential in 81% of patients and incomplete in 19%. Additionally, 64% of patients had adhesions that covered less than 50% of the glans penis, compared to adhesions that covered 50% or more in 36% of patients. After the initial visit, 66% of patients elected the home-care regimen, 89% of which were seen in follow-up visits. Only 36% of patients treated with the home-care regimen had completely or substantially resolved adhesions after a median of 6 months. Results showed a significant difference in success with home care depending on the circumferential extent of adhesions (p < 0.05), with patients whose adhesions covered less than 25% of the glans penis having the greatest likelihood of improvement. Effectiveness of home therapy also varied depending on the severity of adhesions (p < 0.05), with patients whose adhesions covered less than 50% of the glans having a higher chance of success. Most patients that initially chose the home-care regimen (65%) eventually elected to undergo LOA in the office. This study is limited by the retrospective nature, failure to control for potential confounders such as weight and the degree of participation at home, and the time frame after initiation of home therapy, which might have been insufficient for complete resolution of adhesions. DISCUSSION: The conservative management of post-circumcision adhesions is not effective over a 6 month time frame, with the great majority of the patients eventually undergoing LOA. Physicians can classify the severity and extent of adhesions to predict what patients might have more success with conservative management. LOA might be better suited for patients with more severe adhesions.

Selective Binding of Small Molecules to Vibrio cholerae DsbA Offers a Starting Point for the Design of Novel Antibacterials.

DsbA enzymes catalyze oxidative folding of proteins that are secreted into the periplasm of Gram-negative bacteria, and they are indispensable for the virulence of human pathogens such as Vibrio cholerae and Escherichia coli. Therefore, targeting DsbA represents an attractive approach to control bacterial virulence. X-ray crystal structures reveal that DsbA enzymes share a similar fold, however, the hydrophobic groove adjacent to the active site, which is implicated in substrate binding, is shorter and flatter in the structure of V. cholerae DsbA (VcDsbA) compared to E. coli DsbA (EcDsbA). The flat and largely featureless nature of this hydrophobic groove is challenging for the development of small molecule inhibitors. Using fragment-based screening approaches, we have identified a novel small molecule, based on the benzimidazole scaffold, that binds to the hydrophobic groove of oxidized VcDsbA with a KD of 446±10 µM. The same benzimidazole compound has ∼8-fold selectivity for VcDsbA over EcDsbA and binds to oxidized EcDsbA, with KD >3.5 mM. We generated a model of the benzimidazole complex with VcDsbA using NMR data but were unable to determine the structure of the benzimidazole bound EcDsbA using either NMR or X-ray crystallography. Therefore, a structural basis for the observed selectivity is unclear. To better understand ligand binding to these two enzymes we crystallized each of them in complex with a known ligand, the bile salt sodium taurocholate. The crystal structures show that taurocholate adopts different binding poses in complex with VcDsbA and EcDsbA, and reveal the protein-ligand interactions that stabilize the different modes of binding. This work highlights the capacity of fragment-based drug discovery to identify inhibitors of challenging protein targets. In addition, it provides a starting point for development of more potent and specific VcDsbA inhibitors that act through a novel anti-virulence mechanism.

Structural Features of Iperoxo-BQCA Muscarinic Acetylcholine Receptor Hybrid Ligands Determining Subtype Selectivity and Efficacy.

Selective agonists for the human M1 and M4 muscarinic acetylcholine receptors (mAChRs) are attractive candidates for the treatment of cognitive disorders, such as Alzheimer's disease and schizophrenia. Past efforts to optimize a ligand for selective agonism at any one of the M1-M5 mAChR subtypes has proven to be a significant challenge. Recently, research efforts have demonstrated that hybrid ligands may offer a potential solution to the lack of selectivity at mAChRs. In an attempt to design M1 mAChR selective agonists by hybridizing an M1 mAChR selective positive allosteric modulator [1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid] and a potent agonist [(4-[(4,5-dihydro-3-isoxazolyl)oxy]-N,N,N-trimethyl-2-butyn-1-aminium iodide) (iperoxo)], we unexpectedly discovered that these ligands possessed noticeable M2/M4 mAChR selectivity. Evaluation of truncated derivatives of the hybrid ligands at the M1-M5 mAChR subtypes suggests that the allosteric pharmacophore of iperoxo-based mAChR hybrid ligands likely sterically disrupts the allosteric site of the mAChRs, attenuating the efficacy of M1/M3/M5 mAChR responses compared to M2/M4 mAChRs, resulting in a preference for the M2/M4 mAChRs. However, at certain intermediate linker lengths, the effects of this apparent disruption of the allosteric site are diminished, restoring nonselective agonism and suggesting a possible allosteric interaction which is favorable to efficacy at all M1-M5 mAChRs.