Allosteric modulation of GPCR-induced β-arrestin trafficking and signaling by a synthetic intrabody

Agonist-induced phosphorylation of G protein-coupled receptors (GPCRs) is a primary determinant of β-arrestin (βarr) recruitment and trafficking. For several GPCRs, such as the vasopressin type II receptor (V2R), which exhibit high affinity for βarrs, agonist-stimulation first drives the translocation of βarrs to the plasma membrane, followed by endosomal trafficking. We previously found that mutation of a single phosphorylation site in V2R (i.e., V2RT360A) results in near-complete loss of βarr translocation to endosomes although βarrs are robustly recruited to the plasma membrane. Here, we show that a synthetic intrabody referred to as intrabody30 (Ib30), which selectively recognizes an active-like βarr1 conformation, rescues endosomal translocation of βarr1 for V2RT360A. In addition, Ib30 also rescues agonist-induced ERK1/2 MAP kinase activation for V2RT360A to levels similar to that of the wild-type V2R. Molecular dynamics simulations reveal that Ib30 binding promotes active-like conformation in βarr1 with respect to the inter-domain rotation. Interestingly, we also observe that Ib30 enhances the interaction of βarr1 with β2-adaptin, which provides a mechanistic basis for the ability of Ib30 to promote endosomal trafficking of βarr1. Taken together, our data provide a novel mechanism to positively modulate the receptor-transducer-effector axis for GPCRs using intrabodies, which can potentially be integrated in the current paradigm of GPCR-targeted drug discovery.

Marine-derived pipeline anticancer natural products: a review of their pharmacotherapeutic potential and molecular mechanisms

Background Cancer is a complex and most widespread disease and its prevalence is increasing worldwide, more in countries that are witnessing urbanization and rapid industrialization changes. Although tremendous progress has been made, the interest in targeting cancer has grown rapidly every year. This review underscores the importance of preventive and therapeutic strategies. Main text Natural products (NPs) from various sources including plants have always played a crucial role in cancer treatment. In this growing list, numerous unique secondary metabolites from marine sources have added and gaining attention and became potential players in drug discovery and development for various biomedical applications. Many NPs found in nature that normally contain both pharmacological and biological activity employed in pharmaceutical industry predominantly in anticancer pharmaceuticals because of their enormous range of structure entities with unique functional groups that attract and inspire for the creation of several new drug leads through synthetic chemistry. Although terrestrial medicinal plants have been the focus for the development of NPs, however, in the last three decades, marine origins that include invertebrates, plants, algae, and bacteria have unearthed numerous novel pharmaceutical compounds, generally referred as marine NPs and are evolving continuously as discipline in the molecular targeted drug discovery with the inclusion of advanced screening tools which revolutionized and became the component of antitumor modern research. Conclusions This comprehensive review summarizes some important and interesting pipeline marine NPs such as Salinosporamide A, Dolastatin derivatives, Aplidine/plitidepsin (Aplidin®) and Coibamide A, their anticancer properties and describes their mechanisms of action (MoA) with their efficacy and clinical potential as they have attracted interest for potential use in the treatment of various types of cancers.

Unbiased Identification of Extracellular Protein–Protein Interactions for Drug Target and Biologic Drug Discovery

Technological improvements in unbiased screening have accelerated drug target discovery. In particular, membrane-embedded and secreted proteins have gained attention because of their ability to orchestrate intercellular communication. Dysregulation of their extracellular protein–protein interactions (ePPIs) underlies the initiation and progression of many human diseases. Practically, ePPIs are also accessible for modulation by therapeutics since they operate outside of the plasma membrane. Therefore, it is unsurprising that while these proteins make up about 30% of human genes, they encompass the majority of drug targets approved by the FDA. Even so, most secreted and membrane proteins remain uncharacterized in terms of binding partners and cellular functions. To address this, a number of approaches have been developed to overcome challenges associated with membrane protein biology and ePPI discovery. This chapter will cover recent advances that use high-throughput methods to move towards the generation of a comprehensive network of ePPIs in humans for future targeted drug discovery.

Engineering ‘Enzymelink’ for screening lead compounds to inhibit mPGES-1 while maintaining prostacyclin synthase activity

Aim: This study investigated our Enzymelinks, COX-2-10aa-mPGES-1 and COX-2-10aa-PGIS, as cellular cross-screening targets for quick identification of lead compounds to inhibit inflammatory PGE2 biosynthesis while maintaining prostacyclin synthesis. Methods: We integrated virtual and wet cross-screening using Enzymelinks to rapidly identify lead compounds from a large compound library. Results: From 380,000 compounds virtually cross-screened with the Enzymelinks, 1576 compounds were identified and used for wet cross-screening using HEK293 cells that overexpressed individual Enzymelinks as targets. The top 15 lead compounds that inhibited mPGES-1 activity were identified. The top compound that specifically inhibited inflammatory PGE2 biosynthesis alone without affecting COX-2 coupled to PGI2 synthase (PGIS) for PGI2 biosynthesis was obtained. Conclusion: Enzymelink technology could advance cyclooxygenase pathway-targeted drug discovery to a significant degree.

Levosimendan prevents tau pathology by inhibiting disulfide-linked tau oligomerization posing as a promising anti-tau therapeutics

Tau oligomers play critical roles in tau pathology, responsible for neuronal cell death and transmitting the disease in the brain. Accordingly, preventing tau oligomerization becomes an important therapeutic strategy to treat tauopathies including Alzheimer’s disease, however progress has been slow due to difficulties of detecting tau oligomers in cellular context. Toward tau-targeted drug discovery, our group have developed a tau-BiFC platform to monitor and quantify tau oligomerization. By using the tau-BiFC platform, we screened 1,018 compounds in FDA-approved & Passed Phase I drug library, and identified levosimendan as a potent anti-tau agent inhibiting tau oligomerization. 14C-isotope labeling of levosimendan identified that levosimendan covalently bound to tau cysteines, directly inhibiting disulfide-linked tau oligomerization. In addition, levosimendan was able to disassemble tau oligomers into monomers, and rescuing neurons from aggregation states. In comparison, the well-known anti-tau agents, methylene blue (MB) and LMTM, failed to protect neurons from tau-mediated toxicity, generating high-molecular weight tau oligomers. The administration of levosimendan also suppressed tau pathology in the brain, preventing cognitive declines in TauP301L-BiFC transgenic mice. Although careful validation is required, here we present the potential of levosimendan as a disease modifying therapy for tauopathies targeting tau oligomerization.

Rationally derived inhibitors of hepatitis C virus (HCV) p7 channel activity reveal prospect for bimodal antiviral therapy

Since the 1960s, a single class of agent has been licensed targeting virus-encoded ion channels, or ‘viroporins’, contrasting the success of channel blocking drugs in other areas of medicine. Although resistance arose to these prototypic adamantane inhibitors of the influenza A virus (IAV) M2 proton channel, a growing number of clinically and economically important viruses are now recognised to encode essential viroporins providing potential targets for modern drug discovery. We describe the first rationally designed viroporin inhibitor with a comprehensive structure-activity relationship (SAR). This step-change in understanding not only revealed a second biological function for the p7 viroporin from hepatitis C virus (HCV) during virus entry, but also enabled the synthesis of a labelled tool compound that retained biological activity. Hence, p7 inhibitors (p7i) represent a unique class of HCV antiviral targeting both the spread and establishment of infection, as well as a precedent for future viroporin-targeted drug discovery.

Foundational research and NIH funding enabling Emergency Use Authorization of remdesivir for COVID-19

Emergency Use Authorization for remdesivir months after discovery of COVID19 is unprecedented. Typically, decades of research and public sector funding are required to establish the mature body of foundational research requisite for efficient, targeted drug discovery and development. This work quantifies the body of research related to the biological target of remdesivir, RNA-dependent RNA polymerase (RdRp), or parent chemical structure, nucleoside analogs (NcAn), through 2019, as well as NIH funding for this research from 2000 to 2019. There were 6,567 RdRp related publications in PubMed, including 1,263 with NIH support, and 11,073 NcAn-related publications, including 2,319 with NIH support. NIH support for RdRp research comprised 2,203 Project Years with Costs of $1,875 million. NIH support for NcAn research comprised 4,607 Project Years with Costs of $4,612 million. Research Project grants accounted for 63% and 48% of Project Years for RdRp and NcAn respectively, but only 19% and 12% of Project Costs. Analytical modeling of research maturation estimates that RdRp and NcAn research passed an established maturity threshold in 2008 and 1994 respectively. Of 97 investigational compounds targeting RdRp since 1989, the three authorized for use entered clinical trials after both thresholds. This work demonstrates the scale of foundational research on the biological target and parent chemical structure of remdesivir that supported its discovery and development for COVID19. This work identifies $6.5 billion in NIH funding for research leading to remdesivir, underscoring the role of public sector investments in basic research and research infrastructure that underlie new drugs and the response to emergent disease.