Miniaturized peptidomimetics and nano-vesiculation in endothelin types through probable nano-disk formation and structure property relationships of endothelins’ fragments

Endothelins (ETs), which are multi-functional-peptides with potential for antagonist-based-therapy in various physiological-malfunctionings, including cardiovascular, nephrological, oncologic, and diabetic conditions, may produce newer chemical entities and drug leads. The present study deals with molecular-modeling of the ETs’ sub-types, ET-I, II, and III to find the structure property-relationship (SPR) of the ETs, and individual fragments derived from the ET sub-type ET-I. The ETs peptidic tails’ amino acid (AA) sequence’s structural differences and similarities, various dissected fragments of the ET-I, and SPR comparison with the sarafotoxin-6b (SRT-6b), a structurally-related snake-venom, showed points of dissimilarities for their structural specifications, geometric disposition, and physico-chemical properties. The generation of miniaturized (shortened sequence) peptides towards offering peptidomimetic compounds of near- and far-values compared SPR with estimations for log P, hydration energy, and other molecular and quantitative structure activity relationship (QSAR) were based on random and ordered-fragments derived from the original ET-I AA’s sequence, and sequential distance changes in the original ET-I sequence’s chain of 1–21 AA. The feasibility of alternate and bond length parameters-based possible cysteine–cysteine cyclizations, sequence homology, AA’s positional demarcation, and presence/absence of cysteines, homology-based basic non-cysteine and cysteines-AA based cyclization, total structure and fragments end-to-end cyclizations, and geometrical analogy-based miniaturized sequence of the shorter AAs from the original ET-I sequence, together with mutated replacements with naturally constituent AAs of the ETs, and SRT-6 sequences were utilized. The major findings of the fragmented sequences, and sequences at par with the original ETs to provide structures similar to the size, volume and with molecular and electronic properties of electrostatic potential and total charge density distribution, crucial factors in receptor bindings were investigated. The SPRs, molecular properties, and QSAR values were estimated to compare and validate the findings with the known homologous compounds, ET-I, and its known and potent antagonists. The study resulted in leads of smaller and larger sizes of peptide-based compounds which may have prospects as potent antagonist and in future needs their bioactivity evaluations after the synthesis. Moreover, approach to plausible vesiculation of the ETs, and the involved processes and structural requirements, together with the molecular interactions in settling a nano-vesicle of the peptidic structure with a possible mechanism is also suggested.

Wnt Inhibitory Factor 1 Binds to and Inhibits the Activity of Sonic Hedgehog

The hedgehog (Hh) and Wnt pathways, crucial for the embryonic development and stem cell proliferation of Metazoa, have long been known to have similarities that argue for their common evolutionary origin. A surprising additional similarity of the two pathways came with the discovery that WIF1 proteins are involved in the regulation of both the Wnt and Hh pathways. Originally, WIF1 (Wnt Inhibitory Factor 1) was identified as a Wnt antagonist of vertebrates, but subsequent studies have shown that in Drosophila, the WIF1 ortholog serves primarily to control the distribution of Hh. In the present, work we have characterized the interaction of the human WIF1 protein with human sonic hedgehog (Shh) using Surface Plasmon Resonance spectroscopy and reporter assays monitoring the signaling activity of human Shh. Our studies have shown that human WIF1 protein binds human Shh with high affinity and inhibits its signaling activity efficiently. Our observation that the human WIF1 protein is a potent antagonist of human Shh suggests that the known tumor suppressor activity of WIF1 may not be ascribed only to its role as a Wnt inhibitor.

Unique Pharmacological Properties of α-Conotoxin OmIA at α7 nAChRs

OmIA, isolated from Conus omaria venom, is a potent antagonist at α7 nAChRs. We determined the co-crystal structure of OmIA with Lymnae stagnalis acetylcholine binding protein (Ls-AChBP) that identified His5, Val10 and Asn11 as key determinants for the high potency of OmIA at α7 nAChRs. Remarkably, despite a competitive binding mode observed in the co-crystal structure, OmIA and analogues displayed functional insurmountable antagonism at α7 and α3β4 nAChRs, except OmIA analogues having long side chain at position 10 ([V10Q]OmIA and [V10L]OmIA), which were partial insurmountable antagonist at α7 nAChRs in the presence of type II positive allosteric modulators (PAMs). A “two-state, two-step” model was used to explain these observations, with [V10Q]OmIA and [V10L]OmIA co-existing in a fast reversible/surmountable as well as a tight binding/insurmountable state. OmIA and analogues also showed biphasic-inhibition at α7 nAChRs in the presence of PNU120596, with a preference for the high-affinity binding site following prolonged exposure. The molecular basis of binding and complex pharmacological profile of OmIA at α7 nAChRs presented in here expands on the potential of α-conotoxins to probe the pharmacological properties of nAChRs and may help guide the development novel α7 modulators.

The Prostacyclin Analogue, Treprostinil, Used in the Treatment of Pulmonary Arterial Hypertension, is a Potent Antagonist of TREK-1 and TREK-2 Potassium Channels

Pulmonary arterial hypertension (PAH) is an aggressive vascular remodeling disease that carries a high morbidity and mortality rate. Treprostinil (Remodulin) is a stable prostacyclin analogue with potent vasodilatory and anti-proliferative activity, approved by the FDA and WHO as a treatment for PAH. A limitation of this therapy is the severe subcutaneous site pain and other forms of pain experienced by some patients, which can lead to significant non-compliance. TWIK-related potassium channels (TREK-1 and TREK-2) are highly expressed in sensory neurons, where they play a role in regulating sensory neuron excitability. Downregulation, inhibition or mutation of these channels leads to enhanced pain sensitivity. Using whole-cell patch-clamp electrophysiological recordings, we show, for the first time, that treprostinil is a potent antagonist of human TREK-1 and TREK-2 channels but not of TASK-1 channels. An increase in TASK-1 channel current was observed with prolonged incubation, consistent with its therapeutic role in PAH. To investigate treprostinil-induced inhibition of TREK, site-directed mutagenesis of a number of amino acids, identified as important for the action of other regulatory compounds, was carried out. We found that a gain of function mutation of TREK-1 (Y284A) attenuated treprostinil inhibition, while a selective activator of TREK channels, BL-1249, overcame the inhibitory effect of treprostinil. Our data suggests that subcutaneous site pain experienced during treprostinil therapy may result from inhibition of TREK channels near the injection site and that pre-activation of these channels prior to treatment has the potential to alleviate this nociceptive activity.

Action of Carvacrol on Parascaris sp. and Antagonistic Effect on Nicotinic Acetylcholine Receptors

Parascaris sp. is the only ascarid parasitic nematode in equids and one of the most threatening infectious organisms in horses. Only a limited number of compounds are available for treatment of horse helminthiasis, and Parascaris sp. worms have developed resistance to the three major anthelmintic families. In order to overcome the appearance of resistance, there is an urgent need for new therapeutic strategies. The active ingredients of herbal essential oils are potentially effective antiparasitic drugs. Carvacrol is one of the principal chemicals of essential oil from Origanum, Thymus, Coridothymus, Thymbra, Satureja and Lippia herbs. However, the antiparasitic mode of action of carvacrol is poorly understood. Here, the objective of the work was to characterize the activity of carvacrol on Parascaris sp. nicotinic acetylcholine receptor (nAChR) function both in vivo with the use of worm neuromuscular flap preparations and in vitro with two-electrode voltage-clamp electrophysiology on nAChRs expressed in Xenopus oocytes. We developed a neuromuscular contraction assay for Parascaris body flaps and obtained acetylcholine concentration-dependent contraction responses. Strikingly, we observed that 300 µM carvacrol fully and irreversibly abolished Parascaris sp. muscle contractions elicited by acetylcholine. Similarly, carvacrol antagonized acetylcholine-induced currents from both the nicotine-sensitive AChR and the morantel-sensitive AChR subtypes. Thus, we show for the first time that body muscle flap preparation is a tractable approach to investigating the pharmacology of Parascaris sp. neuromuscular system. Our results suggest an intriguing mode of action for carvacrol, being a potent antagonist of muscle nAChRs of Parascaris sp. worms, which may account for its antiparasitic potency.

Assessing the Anti-Inflammatory Activity of the Anxiolytic Drug Buspirone Using CRISPR-Cas9 Gene Editing in LPS-Stimulated BV-2 Microglial Cells

Buspirone is an anxiolytic drug with robust serotonin receptor 1A (Htr1a) agonist activities. However, evidence has demonstrated that this drug also targets the dopamine D3 receptor (Drd3), where it acts as a potent antagonist. In vivo, Drd3 blockade is neuroprotective and reduces inflammation in models of Parkinson’s disease. To test if buspirone also elicited anti-inflammatory activities in vitro, we generated stable Drd3−/− and Htr1a−/− BV2 microglial cell lines using CRISPR-Cas9 technology and then tested the effects of buspirone after lipopolysaccharide (LPS) challenge. We found that LPS exposure had no effect on cell viability, except in Htr1a−/− cells, where viability was reduced (p < 0.001). Drug treatment reduced viability in Drd3−/− cells, but not in WT or Htr1a−/− cells. Buspirone counteracted LPS-induced NO release, NOS2, IL-1β and TNF-α gene expression in WT cells, whereas it exerted limited effects in Drd3−/− or Htr1a−/− microglia. In summary, our findings indicate that buspirone attenuates microglial polarization after LPS challenge. These results also highlight some major effects of Drd3 or Htr1a genetic ablation on microglial biology, raising important questions on the complex role of neurotransmitters in regulating microglia functions.

Paradoxical androgen receptor regulation by small molecule enantiomers

Small molecules that target the androgen receptor (AR) are the mainstay of therapy for lethal castration-resistant prostate cancer (CRPC), yet existing drugs lose their efficacy during continued treatment. This evolution of resistance is due to heterogenous mechanisms which include AR mutations causing the identical drug to activate instead of inhibit the receptor. Understanding in molecular detail the paradoxical phenomenon wherein an AR antagonist is transformed into an agonist by structural mutations in the target receptor is thus of paramount importance. Herein, we describe a reciprocal paradox: opposing antagonist and agonist AR regulation determined uniquely by enantiomeric forms of the same drug structure. The antiandrogen BMS-641988, which has (R)-chirality at C-5 encompasses a previously uncharacterized (S)-stereoisomer that is, surprisingly, a potent agonist of AR, as demonstrated by transcriptional assays supported by cell imaging studies. This duality was reproduced in a series of novel compounds derived from the BMS-641988 scaffold. Coupled with in silico modeling studies, the results inform an AR model that explains the switch from potent antagonist to high-affinity agonist in terms of C-5 substituent steric interactions with helix 12 of the ligand binding site. They imply strategies to overcome AR drug resistance and demonstrate that insufficient enantiopurity in this class of AR antagonist can confound efforts to correlate structure with function.

Identification of a potent antagonist of smoothened in hedgehog signaling

BackgroundHedgehog signaling is essential to the regulation of embryonic development, tissue homeostasis, and stem cell self-renewal, making it a prime target for developing cancer therapeutics. Given the close link between aberrant Hedgehog signaling and cancers, many small molecular compounds have been developed to inhibit Smoothened, a key signal transducer of this pathway, for treating cancer and several such compounds have been approved by the United States Food and Drug Administration (GDC-0449 and LDE-225). However, acquired drug resistance has emerged as an important obstacle to the effective use of these first generation Hedgehog pathway blockers. Thus, new Smoothened inhibitors that can overcome such resistance is an urgent need going forward.ResultsWe established the Smoothened/βarrestin2-GFP high-throughput screening platform based on the mechanistic discovery of Hedgehog signaling pathway, and discovered several active small molecules targeting Smoothened including 0025A. Here we show that 0025A can block the translocation of βarrestin2-GFP to Smoothened, displace Bodipy-cyclopamine binding to wild-type Smoothened or mutant Smoothened-D473H, reduce the accumulation of Smo on primary cilia and the expression of Gli upon Hedgehog stimulation. In addition, we show that 0025A can effectively suppress hair follicle morphogenesis and hair growth in mice.ConclusionsOur results demonstrate that 0025A is a potent antagonist targeting Smoothened wild-type and mutant receptors in the Hedgehog signaling pathway and may provide a new therapy for refractory cancers.