siRNA Therapeutics for the Therapy of COVID-19 and Other Coronaviruses.

The ongoing pandemic of global concern has killed about three million humans and affected around 151 million people worldwide, as of April 30, 2021. Although recently approved vaccines for COVID-19 are engendering hope, finding new ways to cure the viral pandemic is still a quest for researchers worldwide. Major pandemics in history have been of viral origin, such as SARS, MERS, H1NI, Spanish flu, and so on. A larger emphasis has been on discovering potential vaccines, novel antiviral drugs, and agents that can mitigate the viral infection symptoms; however, a relatively new area, RNA interference (RNAi), has proven effective as an antiviral agent. The RNAi phenomenon has been largely exploited to cure cancer, neurodegenerative diseases, and some rare diseases. The U.S. Food and Drug Administration has recently approved three siRNA products for human use that garner significant hope in siRNA therapeutics for coronaviruses. There have been some commentaries and communications addressing this area. We have summarized and illustrated the significance and the potential of the siRNA therapeutics available as of April 30, 2021 to combat the ongoing viral pandemic and the emerging new variants such as B.1.1.7 and B.1.351. Numerous successful in vitro studies and several investigations to address the clinical application of siRNA therapeutics provide great hope in this field. This seminal Review describes the significance of siRNA-based therapy to treat diverse viral infections in addition to the current coronavirus challenge. In addition, we have thoroughly reviewed the patents approved for coronaviruses, the major challenges in siRNA therapy, and the potential approaches to address them, followed by innovation and prospects.

Use of constitutive G protein-coupled receptor activity for drug discovery.

This article describes the behavior of transiently transfected human receptors into melanophores and the potential use of constitutive receptor activity to screen for new drug entities. Specifically, transient transfection of melanophores with different concentrations of receptor cDNA presumably leads to increased levels of receptor expression. This leads to an increased response to agonists (both maxima and potency) and, in some cases, an agonist-independent constitutive receptor activity. Transfections with increasing concentrations of the G(s) protein-coupled human calcitonin receptor type 2 (hCTR2) cDNA produced sufficient levels of constitutively activated receptor to cause elevated basal cellular responses. This was observed as a decrease in the transmittance of light through melanophores (consistent with G(s) protein activation) and increased response to human calcitonin. The receptor-mediated nature of this response was confirmed by its reversal with the hCTR2 peptide inverse agonist AC512. A collection of ligands for hCTR2 either increased or decreased constitutive hCTR2 activity, suggesting that the constitutive system was a sensitive discriminator of positive and negative ligand efficacy. Similar results were obtained with G(i)-protein-coupled receptors. Transient transfection of NPY1, NPY2, NPY4, CXCR4, and CCR5 cDNA produced increased light transmittance through melanophores (consistent with G(i)-protein activation). NPY1 cDNA produced little constitutive response on transfection, whereas maximal levels of constitutive activity ranging from 30 to 45% were observed for the other G(i)-protein-coupled receptors. Responses to agonists for these receptors increased (both maxima and potency) with increasing cDNA transfection. The receptor/G(i)-protein nature of both the constitutive and agonist-mediated responses was confirmed by elimination with pertussis toxin pretreatment. These data are discussed in terms of the theoretical aspects of constitutive receptor activity and the applicability of this approach for the general screening of G protein-coupled orphan receptors.

Constitutive receptor systems for drug discovery.

This paper discusses the use of constitutively active G-protein-coupled receptor systems for drug discovery. Specifically, the ternary complex model is used to define the two major theoretical advantages of constitutive receptor screening-namely, the ability to detect antagonists as well as agonists directly and the fact that constitutive systems are more sensitive to agonists. In experimental studies, transient transfection of Chinese hamster ovary cyclic AMP response element (CRE) luciferase reporter cells with cDNA for human parathyroid hormone receptor, glucagon receptor, and glucagon-like peptide (GLP-1) receptor showed cDNA concentration-dependent constitutive activity with parathyroid hormone (PTH-1) and glucagon. In contrast, no constitutive activity was observed for GLP-1 receptor, yet responses to GLP-1 indicated that receptor expression had taken place. In another functional system, Xenopus laevi melanophores transfected with cDNA for human calcitonin receptor showed constitutive activity. Nine ligands for the calcitonin receptor either increased or decreased constitutive activity in this assay. The sensitivity of the system to human calcitonin increased with increasing constitutive activity. These data indicate that, for those receptors which naturally produce constitutive activity, screening in this mode could be advantageous over other methods.

Expression cloning and receptor pharmacology of human calcitonin receptors from MCF-7 cells and their relationship to amylin receptors.

Human breast cell carcinoma MCF-7 cells were found to bind 125I-labeled rat amylin (rAmylin) and the peptide amylin antagonist radioligand 125I-AC512 with high affinity. This high affinity binding possessed characteristics unique to the already defined high affinity binding site for amylin in the rat nucleus accumbens [Mol. Pharmacol. 44:493-497 (1993); J. Pharmacol. Exp. Ther. 270:779-787 (1994); Eur. J. Pharmacol. 262:133-141 (1994)]. To further define this receptor, we report results of expression cloning studies from an MCF-7 cell library. We isolated two variants of a seven-transmembrane receptor that were identical to two previously described human calcitonin receptors (hCTR1 and hCTR2). These receptors were characterized by expression in different surrogate host cell systems. Transient expression of hCTR1 in COS cells yielded membranes that bound 125I-AC512 and 125I-salmon calcitonin with high affinity, but no high affinity binding was observed with 125I-human calcitonin (hCAL) or 125I-rAmylin. Stable expression of hCTR1 in HEK 293 cells produced similar data. In contrast, expression of hCTR2 in COS cells yielded membranes that bound 125I-AC512, 125I-hCAL, and 125I-rAmylin with high affinity. The agonists 125I-hCAL and 125I-rAmylin bound 65% and 1.5%, respectively, of the sites bound by the antagonist radioligand 125I-AC512 in this expression system. This pattern of binding was repeated in HEK 293 cells stably transfected with hCTR2 (125I-hCAL = 24.8% Bmax, 125I-rAmylin = 8% Bmax). In both expression systems, the agonists hCAL and rAmylin were much more potent in displacing their radioligand counterparts than was the antagonist radioligand 125I-AC512. For example, the pKi value for displacement of 125I-AC512 by rAmylin was 7.2 in HEK 293 cells but rose to 9.1 when displacing 125I-rAmylin. Finally, hCTR2 was expressed in baculovirus-infected Ti ni cells. In this system, only specific binding to the antagonist 125I-AC512 and agonist 125I-hCAL was observed; no binding to 125I-rAmylin could be detected. These data are discussed in terms of two working hypotheses. The first is that amylin is a weak agonist for hCTR2 and that this receptor is unrelated to the amylin receptor found in this cell line. The second is that hCTR2 couples to different G proteins for calcitonin and amylin function in different cells. At present, these data cannot be used to disprove conclusively either hypothesis.

A degeneration study of some habenular efferents to the midbrain in a wallaby.

In this study, specimens of both sexes of a Western Australian wallaby (Steonix brachyurus) had surgical or electrolytic lesions made in the habenular complex unilaterally. Thirty-five-micron sections, cut coronally and parasagittally, were stained by the Nauta-Gygax ('54) or the Fink-Heimer ('67) technique to demonstrate degenerating fibers. Degeneration was seen in the habenular commissure and bilaterally in the habenula and fasciculus retroflexus, but was most extensive ipsilaterally. Habenular fibers destined for the tegmentum and central gray passed caudally into the midbrain in a continuous fan-like array which extended from the central gray dorsally to the interpeduncular nucleus ventrally. In their posterior course some fibers crossed the midline with the decussating cerebellar fibers. The majority of fibers, with those in the fasciculus retroflexus which passed ventral to the decussation of the superior cerebellar peduncle, turned dorsally. They passed mostly just lateral to the midline nuclei and into the central gray where degeneration was seen throughout the entire extent. Terminal degeneration was seen in the interpeduncular nucleus primarily in the dorsal and posterior parts, in the ventral tegmental nucleus and throughout the central gray, but was more densely clustered in the region of the dorsal tegmental nucleus.