Generation of KS-133 as a Novel Bicyclic Peptide with a Potent and Selective VIPR2 Antagonist Activity that Counteracts Cognitive Decline in a Mouse Model of Psychiatric Disorders

Worldwide, more than 20 million people suffer from schizophrenia, but effective and definitive new therapeutic drugs/treatments have not been established. Vasoactive intestinal peptide receptor 2 (VIPR2) might be an attractive drug target for the treatment of schizophrenia because both preclinical and clinical studies have demonstrated a strong link between high expression/overactivation of VIPR2 and schizophrenia. Nevertheless, VIPR2-targeting drugs are not yet available. VIPR2 is a class-B G protein-coupled receptor that possesses high structural homology to its subtypes, vasoactive intestinal peptide receptor 1 (VIPR1) and pituitary adenylate cyclase-activating polypeptide type-1 receptor (PAC1). These biological and structural properties have made it difficult to discover small molecule drugs against VIPR2. In 2018, cyclic peptide VIpep-3, a VIPR2-selective antagonist, was reported. The aim of this study was to generate a VIpep-3 derivative for in vivo experiments. After amino acid substitution and structure optimization, we successfully generated KS-133 with 1) a VIPR2-selective and potent antagonistic activity, 2) at least 24 h of stability in plasma, and 3) in vivo pharmacological efficacies in a mouse model of psychiatric disorders through early postnatal activation of VIPR2. To the best of our knowledge, this is the first report of a VIPR2-selective antagonistic peptide that counteracts cognitive decline, a central feature of schizophrenia. KS-133 may contribute to studies and development of novel schizophrenia therapeutic drugs that target VIPR2.

Voided urine test to diagnose prostate cancer: Preliminary report

Objectives: Prostate cancer (PCa) is a common malignancy affecting elderly male. At present, PCa is estimated using serum prostate-specific antigen (PSA). Prostate biopsy remains the gold standard to confirm the diagnosis of PCa. In this preliminary study, we have assessed the feasibility of detecting PCa using voided urine by targeting the genomic vasoactive intestinal peptide receptor (VPAC) expressed on malignant PCa cells. Material and Methods: Patients ≥40 years old, with no lower urinary tract symptoms (LUTS) and serum PSA levels of <1.6 ng/mL formed the control group and patients ≥40 years old, with LUTS and serum PSA >2.6 ng/ mL formed the study group. Patients were advised to give the first 50 mL of voided urine sample for the detection of malignant markers by targeting the VPAC. The results of histopathological studies were then compared to the results of urine biomarker. Results: The study revealed absence of malignant markers in 75 patients (control group). In the study group, all the 33 patients with adenocarcinoma were positive for malignant markers in the biomarker study and absence of malignant markers in the 32 patients with benign histology. The results of the biomarker studies and histopathology were consistent with each other. Conclusion: This preliminary study validates our belief that patients with PCa do shed malignant cells in the urine which can be identified by targeting the VPAC. The investigation is easy and our data appear to be highly encouraging and further serve as a simple, reliable, and a non-invasive tool in the detection of PCa.

Tamoxifen-inducible cardiac-specific Cre transgenic mouse using VIPR2 intron

Genetically engineered mouse models through gene deletion are useful tools for analyzing gene function. To delete a gene in a certain tissue temporally, tissue-specific and tamoxifen-inducible Cre transgenic mice are generally used. Here, we generated transgenic mouse with cardiac-specific expression of Cre recombinase fused to a mutant estrogen ligand-binding domain (ERT2) on both N-terminal and C-terminal under the regulatory region of human vasoactive intestinal peptide receptor 2 (VIPR2) intron and Hsp68 promoter (VIPR2-ERT2CreERT2). In VIPR2-ERT2CreERT2 transgenic mice, mRNA for Cre gene was highly expressed in the heart. To further reveal heart-specific Cre expression, VIPR2-ERT2CreERT2 mice mated with ROSA26-lacZ reporter mice were examined by X-gal staining. Results of X-gal staining revealed that Cre-dependent recombination occurred only in the heart after treatment with tamoxifen. Taken together, these results demonstrate that VIPR2-ERT2CreERT2 transgenic mouse is a useful model to unveil a specific gene function in the heart.

The myopia susceptibility locus vasoactive intestinal peptide receptor 2 (VIPR2) contains variants with opposite effects

Myopia is the commonest eye disorder in the world. High myopes are predisposed to ocular pathologies. The vasoactive intestinal peptide receptor 2 (VIPR2) gene was identified as a myopia susceptibility locus by our group and another group. We continued to fine-map this locus. A case-control study was performed in 4 sequential stages with a total of 941 highly myopic subjects and 846 control subjects, all unrelated Chinese. Stage 1 experimentally genotyped 64.4% of the entire cohort for 152 single-nucleotide polymorphisms (SNPs) and Stage 2 the remaining subjects for 21 SNPs. Stage 3 combined the genotypes for 21 SNPs for the entire cohort, and identified one group of high-risk haplotypes and one group of protective haplotypes significantly associated with high myopia. Stage 4 imputed genotypes for variants in the VIPR2 region and identified two independent groups of variants: one group with high-risk minor alleles and another with protective minor alleles. Variants within each group were generally in strong linkage disequilibrium among themselves while high-risk variants were in linkage equilibrium with protective variants. Therefore, the VIPR2 locus seems to contain variants with opposite effects. This is the first study that has examined the genetic architecture of a myopia susceptibility locus in detail.

A Molecular Dynamics Study of Vasoactive Intestinal Peptide Receptor 1 and the Basis of Its Therapeutic Antagonism

Vasoactive intestinal peptide receptor 1 (VPAC1) is a member of a secretin-like subfamily of G protein-coupled receptors. Its endogenous neuropeptide (VIP), secreted by neurons and immune cells, modulates various physiological functions such as exocrine and endocrine secretions, immune response, smooth muscles relaxation, vasodilation, and fetal development. As a drug target, VPAC1 has been selected for therapy of inflammatory diseases but drug discovery is still hampered by lack of its crystal structure. In this study we presented the homology model of this receptor constructed with the well-known web service GPCRM. The VPAC1 model is composed of extracellular and transmembrane domains that form a complex with an endogenous hormone VIP. Using the homology model of VPAC1 the mechanism of action of potential drug candidates for VPAC1 was described. Only two series of small-molecule antagonists of confirmed biological activity for VPAC1 have been described thus far. Molecular docking and a series of molecular dynamics simulations were performed to elucidate their binding to VPAC1 and resulting antagonist effect. The presented work provides the basis for the possible binding mode of VPAC1 antagonists and determinants of their molecular recognition in the context of other class B GPCRs. Until the crystal structure of VPAC1 will be released, the presented homology model of VPAC1 can serve as a scaffold for drug discovery studies and is available from the author upon request.

Impaired neonatal cardiorespiratory responses to hypoxia in mice lacking PAC1 or VPAC2 receptors

The stress peptide pituitary adenylate cyclase activating polypeptide (PACAP) and its specific receptor PACAP type 1 receptor (PAC1) have been implicated in sudden infant death syndrome (SIDS). PACAP is also critical to the neonatal cardiorespiratory response to homeostatic stressors identified in SIDS, including hypoxia. However, which of PACAP’s three receptors, PAC1, vasoactive intestinal peptide receptor type 1 (VPAC1), and/or vasoactive intestinal peptide receptor type 2 (VPAC2), are involved is unknown. In this study, we hypothesized that PAC1, but not VPAC2, is involved in mediating the cardiorespiratory response to hypoxia during neonatal development. To test this hypothesis, head-out plethysmography and surface ECG electrodes were used to assess the cardiorespiratory variables of unanesthetized postnatal day 4 PAC1 and VPAC2-knockout (KO) and wild-type (WT) mice in response to a 10% hypoxic challenge. Our results demonstrate that compared with WT pups, the early and late hypoxic rate of expired CO2 (V̇co2), V̇co2 and ventilatory responses were blunted in PAC1-KO neonates, and during the posthypoxic period, minute ventilation (V̇e), V̇co2 and heart rate were increased, while the increase in apneas normally associated with the posthypoxic period was reduced. Consistent with impaired cardiorespiratory control in these animals, the V̇e/V̇co2 slope was reduced in PAC1-KO pups, suggesting that breathing was inappropriately matched to metabolism. In contrast, VPAC2-KO pups exhibited elevated heart rate variability during hypoxia compared with WT littermates, but the effects of the VPAC2-KO genotype on breathing were minimal. These findings suggest that PAC1 plays the principal role in mediating the cardiorespiratory effects of PACAP in response to hypoxic stress during neonatal development and that defective PACAP signaling via PAC1 may contribute to the pathogenesis of SIDS.