Connexons Coupling to Gap Junction Channel: Potential Role for Extracellular Protein Stabilization Centers

Connexin (Cx) proteins establish intercellular gap junction channels (Cx GJCs) through coupling of two apposed hexameric Cx hemichannels (Cx HCs, connexons). Pre- and post-GJ interfaces consist of extracellular EL1 and EL2 loops, each with three conserved cysteines. Previously, we reported that known peptide inhibitors, mimicking a variety of Cx43 sequences, appear non-selective when binding to homomeric Cx43 vs. Cx36 GJC homology model subtypes. In pursuit of finding potentially Cx subtype-specific inhibitors of connexon-connexon coupling, we aimed at to understand better how the GJ interface is formed. Here we report on the discovery of Cx GJC subtype-specific protein stabilization centers (SCs) featuring GJ interface architecture. First, the Cx43 GJC homology model, embedded in two opposed membrane bilayers, has been devised. Next, we endorsed the fluctuation dynamics of SCs of the interface domain of Cx43 GJC by applying standard molecular dynamics under open and closed cystine disulfide bond (CS-SC) preconditions. The simulations confirmed the major role of of the unique trans-GJ SC pattern comprising conserved (55N, 56T) and non-conserved (57Q) residues of the apposed EL1 loops in the stabilization of the GJC complex. Importantly, clusters of SC patterns residing close to the GJ interface domain appear to orient the interface formation via the numerous SCs between EL1 and EL2. These include central 54CS-S198C or 61CS-S192C contacts with residues 53R, 54C, 55N, 197D, 199F or 64V, 191P, respectively. In addition, we revealed that GJC interface formation is favoured when the psi dihedral angle of the nearby 193P residue is stable around 180° and the interface SCs disappear when this angle moves to the 0° to −45° range. The potential of the association of non-conserved residues with SC motifs in connexon-connexon coupling makes the development of Cx subtype-specific inhibitors viable.

Discovery and synthesis of hydroxy-L-proline based blockers of the neutral amino acid transporters SLC1A4 (ASCT1) and SLC1A5 (ASCT2).

The conformationally restricted heterocycle hydroxy-ʟ-proline is a versatile scaffold for the synthesis of diverse multi-functionalized pyrrolidines for probing the ligand binding sites of biological targets. With the goal to develop new inhibitors of the widely expressed amino acid transporters SLC1A4 and SLC1A5 (also known as ASCT1 and ASCT2), we synthesized and functionally screened a series of hydroxy-ʟ-proline derivatives or 'prolinols' using electrophysiological and radio-labeled uptake assays on amino acid transporters from the SLC1, SLC7, and SLC38 solute carrier families. We identified a number of synthetic prolinols that act as selective high-affinity inhibitors of the SLC1 functional subfamily comprising the neutral amino acid transporters SLC1A4 and SLC1A5. The active and inactive prolinols were computationally docked into a threaded homology model and analyzed with respect to predicted molecular orientation and observed pharmacological activity. The series of hydroxy-L-proline derivatives identified here represents a new class of potential agents to pharmacologically modulate SLC1A4 and SLC1A5, amino acid exchangers that play important roles in a wide range of physiological and pathophysiological processes.

A small molecule reveals role of insulin receptor-insulin like growth factor-1 receptor heterodimers

The insulin receptor (IR) and insulin like growth factor-1 receptor (IGF-1R) are heterodimers consisting of 2 extracellular α-subunits and 2 transmembrane β-subunits. IR α/β and IGF-1R α/β hemi-receptors can heterodimerize to form hybrids composed of one IR α/β and one IGF-1R α/β. Widely distributed in mammalian tissues, in contrast to IR and IGF-1R the physiological function of hybrids is unclear. To identify tool compounds that inhibit hybrid formation we performed a high-throughput small molecule screen based on a homology model of hybrid structure. Our studies unveil a first in class quinoline-containing heterocyclic small molecule that reduces hybrids by >50% in human umbilical vein endothelial cells (HUVECs) with no effect on IR or IGF-1R. Downstream of IR and IGF-1R our small molecule led to reduced expression of the negative regulatory p85α subunit of phosphatidylinositol 3-kinase, an increase in phosphorylation of its downstream target Akt and enhanced insulin and shear-induced phosphorylation of Akt. We show that hybrids have a role in human EC physiology distinct from IR and IGF1R.

Structure-based design of a chemical probe set for the 5-HT5A serotonin receptor

ABSTRACTThe 5-HT5A receptor (5-HT5AR), for which no selective agonists and only a few antagonists exist, remains the least understood serotonin (5-HT) receptor. A single commercial antagonist (SB-699551) has been widely used to investigate central nervous system (CNS) 5-HT5AR function in neurological disorders, including pain. However, because SB-699551 has affinity for many 5-HTRs, lacks inactive property-matched controls, and has assay interference concerns, it has liabilities as a chemical probe. To better illuminate 5-HT5AR function, we developed a probe set through iterative rounds of molecular docking, pharmacological testing, and optimization. Docking over six million lead-like molecules against a 5-HT5AR homology model identified five mid-μM ligand starting points with unique scaffolds. Over multiple rounds of structure-based design and testing, a new quinoline scaffold with high affinity and enhanced selectivity for the 5-HT5AR was developed, leading to UCSF678, a 42 nM arrestin-biased partial agonist at the 5-HT5AR with a much more restricted off-target profile and decreased assay liabilities vs. SB-699551. Site-directed mutagenesis supported the docked pose of UCSF678, which was also consistent with recent published 5-HTR structures. Surprisingly, property-matched analogs of UCSF678 that were either inactive across 5-HTRs or retained affinity for UCSF678’s off-targets revealed that 5-HT5AR engagement is nonessential for alleviating pain in a mouse model, contrary to previous studies using less-selective ligands. Relative to SB-699551, these molecules constitute a well-characterized and more selective probe set with which to study the function of the 5-HT5A receptor. Table of Contents Graphic

Substituted Aryl Benzylamines as Potent and Selective Inhibitors of 17β-Hydroxysteroid Dehydrogenase Type 3

17β-Hydroxysteroid dehydrogenase type 3 (17β-HSD3) is expressed at high levels in testes and seminal vesicles; it is also present in prostate tissue and involved in gonadal and non-gonadal testosterone biosynthesis. The enzyme is membrane-bound, and a crystal structure is not yet available. Selective aryl benzylamine-based inhibitors were designed and synthesised as potential agents for prostate cancer therapeutics through structure-based design, using a previously built homology model with docking studies. Potent, selective, low nanomolar IC50 17β-HSD3 inhibitors were discovered using N-(2-([2-(4-chlorophenoxy)phenylamino]methyl)phenyl)acetamide (1). The most potent compounds have IC50 values of approximately 75 nM. Compound 29, N-[2-(1-Acetylpiperidin-4-ylamino)benzyl]-N-[2-(4-chlorophenoxy)phenyl]acetamide, has an IC50 of 76 nM, while compound 30, N-(2-(1-[2-(4-chlorophenoxy)-phenylamino]ethyl)phenyl)acetamide, has an IC50 of 74 nM. Racemic C-allyl derivative 26 (IC50 of 520 nM) was easily formed from 1 in good yield and, to determine binding directionality, its enantiomers were separated by chiral chromatography. Absolute configuration was determined using single crystal X-ray crystallography. Only the S-(+)-enantiomer (32) was active with an IC50 of 370 nM. Binding directionality was predictable through our in silico docking studies, giving confidence to our model. Importantly, all novel inhibitors are selective over the type 2 isozyme of 17β-HSD2 and show <20% inhibition when tested at 10 µM. Lead compounds from this series are worthy of further optimisation and development as inhibitors of testosterone production by 17β-HSD3 and as inhibitors of prostate cancer cell growth.

Drug Repositioning For Allosteric Modulation of VIP and PACAP Receptors

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are two neuropeptides that contribute to the regulation of intestinal motility and secretion, exocrine and endocrine secretions, and homeostasis of the immune system. Their biological effects are mediated by three receptors named VPAC1, VPAC2 and PAC1 that belong to class B GPCRs. VIP and PACAP receptors have been identified as potential therapeutic targets for the treatment of chronic inflammation, neurodegenerative diseases and cancer. However, pharmacological use of endogenous ligands for these receptors is limited by their lack of specificity (PACAP binds with high affinity to VPAC1, VPAC2 and PAC1 receptors while VIP recognizes both VPAC1 and VPAC2 receptors), their poor oral bioavailability (VIP and PACAP are 27- to 38-amino acid peptides) and their short half-life. Therefore, the development of non-peptidic small molecules or specific stabilized peptidic ligands is of high interest. Structural similarities between VIP and PACAP receptors are major causes of difficulties in the design of efficient and selective compounds that could be used as therapeutics. In this study we performed structure-based virtual screening against the subset of the ZINC15 drug library. This drug repositioning screen provided new applications for a known drug: ticagrelor, a P2Y12 purinergic receptor antagonist. Ticagrelor inhibits both VPAC1 and VPAC2 receptors which was confirmed in VIP-binding and calcium mobilization assays. A following analysis of detailed ticagrelor binding modes to all three VIP and PACAP receptors with molecular dynamics revealed its allosteric mechanism of action. Using a validated homology model of inactive VPAC1 and a recently released cryo-EM structure of active VPAC1 we described how ticagrelor could block conformational changes in the region of ‘tyrosine toggle switch’ required for the receptor activation. We also discuss possible modifications of ticagrelor comparing other P2Y12 antagonist – cangrelor, closely related to ticagrelor but not active for VPAC1/VPAC2. This comparison with inactive cangrelor could lead to further improvement of the ticagrelor activity and selectivity for VIP and PACAP receptor sub-types.

A Missense Mutation (R723H) in the Head Motor Domain of β-MYH7 gene in an Indian HCM Patient and Phenotypic Plasticity

Mutations in the β-MYH7 gene are one of the major causes that lead to cardiomyopathies. However, to differentiate a causative nsSNP and its impact on protein structure remains a major challenge. In the present study, we detected a missense mutation Arg723His in the head motor domain of β-MYH7 in a HCM patient, and it was absent in 207 healthy individuals. The mutant (R723H) has been found to alter an evolutionarily conserved amino acid. In addition, the mutant (R723H) was predicted pathogenic by Polyphen-2 and SIFT bioinformatic tools. Further, the superimposed 3D structure of the mutant (p.His723 homology model) with native (p. Arg723) displayed the root means square deviation (RMSD) of ~3.38A0. We know that the non-covalent interactions such as hydrophobic, electrostatic, Van der Waals, and hydrogen bonds between amino acids are at the heart of stabilizing protein structures. Here, we demonstrated how the mutant (p.His723) has disrupted a critical non-covalent interactions network at the mutation site and may contribute to the disease phenotype. Hence, our findings in the future could pave the way for developing small molecular modulators or myosin-targeted therapies for heart failure.

Chalcones identify cTXNPx as a potential antileishmanial drug target

With current drug treatments failing due to toxicity, low efficacy and resistance; leishmaniasis is a major global health challenge that desperately needs new validated drug targets. Inspired by activity of the natural chalcone 2’,6’-dihydroxy-4’-methoxychalcone (DMC), the nitro-analogue, 3-nitro-2’,4’,6’- trimethoxychalcone (NAT22, 1c) was identified as potent broad spectrum antileishmanial drug lead. Structural modification provided an alkyne containing chemical probe that labelled a protein within the parasite that was confirmed as cytosolic tryparedoxin peroxidase (cTXNPx). Crucially, labelling is observed in both promastigote and intramacrophage amastigote life forms, with no evidence of host macrophage toxicity. Incubation of the chalcone in the parasite leads to ROS accumulation and parasite death. Deletion of cTXNPx, by CRISPR-Cas9, dramatically impacts upon the parasite phenotype and reduces the antileishmanial activity of the chalcone analogue. Molecular docking studies with a homology model of in-silico cTXNPx suggest that the chalcone is able to bind in the putative active site hindering access to the crucial cysteine residue. Collectively, this work identifies cTXNPx as an important target for antileishmanial chalcones.

Fragment-Based Virtual Screening to Discover Potential Plasmodium PI4KIIIβ Ligands

Plasmodium species that cause malaria, a disease responsible for about half a million deaths per annum despite concerted efforts to combat it. The causative agent depends on type III beta phosphatidylinositol 4-kinase (PPI4K) during the development of merozoite. PPI4K is the only clinically validated Plasmodium kinase so far and its inhibitors are effective both in vitro and in vivo. In this work, a small library of ~22 000 fragments was virtually screened using PPI4K homology model to discover potential ligands of the enzyme. 16 virtual hits were selected based on &le; -9.0 kcal/mol binding energy cut off and were subjected to similarity and substructure searching after they had passed PAINS screening. The derivatives obtained showed improved binding energies, which ranged from -10.00 to -13.80 kcal/mol. Moreover, the topmost ranking compound 31, with interesting drug-like quality was stable within the protein&rsquo;s binding cavity during the 10 ns molecular dynamics simulation period. In addition, analysis of its binding pose revealed some unique binding interactions with PPI4K active site residues as the basis for the observed improved binding affinity. Overall, compound 31 appears to be a viable starting point for the development of PPI4K inhibitors with antimalarial activity.