The influence of several nutritional supplements on the rational use of cabozantinib.

To promote the rational use of cabozantinib (CBZ), this paper studied the influence of several nutritional supplements on the interaction between CBZ and bovine serum albumin (BSA), an appropriate alternative model for human serum albumin (HSA) that is one of the important transporter proteins in plasma, by fluorescence spectroscopy and UV-vis spectroscopy. The results showed that CBZ could quench the fluorescence of BSA via a dynamic-static quenching process, and the six nutritional supplements did not change the quenching mode of BSA by CBZ. However, all of them could reduce the binding constant of the CBZ-BSA system at 293 K and increase the polarity around tryptophan residues. Among them, nicotinamide and vitamin B12 (VB12 ) had a greater effect on the binding constants of the CBZ-BSA system. In the meantime, the thermodynamic parameters of the CBZ-BSA system were examined, indicating that the interaction of CBZ with BSA was spontaneous and dominated by hydrophobic forces. Further research discovered that the combining of CBZ with BSA was primarily located within Site I of BSA, and the binding distance r was 2.48 nm. Consequently, while taking CBZ, patients should use VB12 and nicotinamide carefully, which may interfere with the transport of drugs.

There is nothing exempt from the peril of mutation - The Omicron spike.

The 2019 corona virus disease (COVID-19) has caused a global chaos, where a novel Omicron variant has challenged the healthcare system, followed by which it has been referred to as a variant of concern (VOC) by the World Health Organization (WHO), owing to its alarming transmission and infectivity rate. The large number of mutations in the receptor binding domain (RBD) of the spike protein is responsible for strengthening of the spike-angiotensin-converting enzyme 2 (ACE2) interaction, thereby explaining the elevated threat. This is supplemented by enhanced resistance of the variant towards pre-existing antibodies approved for the COVID-19 therapy. The manuscript brings into light failure of existing therapies to provide the desired effect, however simultaneously discussing the novel possibilities on the verge of establishing suitable treatment portfolio. The authors entail the risks associated with omicron resistance against antibodies and vaccine ineffectiveness on one side, and novel approaches and targets - kinase inhibitors, viral protease inhibitors, phytoconstituents, entry pathways - on the other. The manuscript aims to provide a holistic picture about the Omicron variant, by providing comprehensive discussions related to multiple aspects of the mutated spike variant, which might aid the global researchers and healthcare experts in finding an optimised solution to this pandemic.

Lyophyllin, a Mushroom Protein from the Peptidase M35 Superfamily Is an RNA N-Glycosidase.

Ribosome-inactivating proteins (RIPs) hydrolyze the N-glycosidic bond and depurinate a specific adenine residue (A-4324 in rat 28S ribosomal RNA, rRNA) in the conserved α-sarcin/ricin loop (α-SRL) of rRNA. In this study, we have purified and characterized lyophyllin, an unconventional RIP from Lyophyllum shimeji, an edible mushroom. The protein resembles peptidase M35 domain of peptidyl-Lys metalloendopeptidases. Nevertheless, protein either from the mushroom or in recombinant form possessed N-glycosidase and protein synthesis inhibitory activities. A homology model of lyophyllin was constructed. It was found that the zinc binding pocket of this protein resembles the catalytic cleft of a classical RIP, with key amino acids that interact with the adenine substrate in the appropriate positions. Mutational studies showed that E122 may play a role in stabilizing the positively charged oxocarbenium ion and H121 for protonating N-3 of adenine. The tyrosine residues Y137 and Y104 may be used for stacking the target adenine ring. This work first shows a protein in the peptidase M35 superfamily based on conserved domain search possessing N-glycosidase activity.

Deciphering the AChE-binding mechanism with multifunctional tricyclic coumarin anti-Alzheimer's agents using biophysical and bioinformatics approaches and evaluation of their modulating effect on Amyloidogenic peptide assembly.

Investigating the drug-AChE binding mechanism is vital in understanding its cogent use in medical practice against Alzheimer's disease (AD). The production and accumulation of oligomers of ß-amyloid is a central event in the neuropathology of AD. Beside the inhibition of assembly process, modulation of the aggregation process of these proteins towards minimally toxic pathways may be a possible therapeutic strategy for AD. Hence, the present study aims to examine the effect of multifunctional fused tricyclic 7-hydroxy 4-methyl coumarin analogs (HMC1-5) on the self-induced aggregation of ß-amyloid using Thioflavin T (ThT) assay, scanning electron microscopic study, AlamarBlue and immune blotting assays and also the binding mechanism with AChE by fluorescence emission, conformational, molecular docking and molecular dynamic simulation studies under physiological pH 7.4. The ThT assay, FE-SEM study, cell line and western blots establish that the HMC1-5 molecules could irreversibly disrupt preformed Aß42 fibrils, accelerate the aggregates into micro size co-assembled structures, and effectively eliminate the cytotoxicity of Aß1-42. Fluorescence emission studies indicating a strong binding affinity between HMC1-5 and AChE with the binding constants of 1.04 × 105, 3.57 × 104, 1.97 × 104, 3.07 × 104 and 2.95 × 104 M-1, respectively and binding sites number found to be 1. CD studies disclosed a partial unfolding in the secondary structure of AChE upon binding with HMC1-5. Docking analysis inferred that the HMC1-5 were bound through hydrophobic and hydrophilic interactions to the AChE active site. Molecular dynamics simulations emphasized the stability of AChE-HMC1-5 complexes throughout the 100 ns simulations, and the local conformational changes of the residues of AChE validate the stability of complexes. These results provide new and unique complementary approach for modulating the biological effects of the Aß aggregates by coumarin analogs and new insights for further in vivo investigations as novel anti AD agents.

Drug affinity responsive target stability (DARTS) accelerated small molecules target discovery: Principles and application.

Drug affinity responsive target stability (DARTS) is a novel target discovery approach and is particularly adept at screening small molecule (SM) targets without requiring any structural modifications. The DARTS method is capable of revealing drug-target interactions from cells or tissues by tracking changes in the stability of proteins acting as receptors of bioactive SMs. Due to its simple operation and high efficiency, the DARTS method has been applied to uncover the drug-action mechanism. This review summarized analytical principles, protocols, validation approaches, applications, and challenges involved in the DARTS method. Due to the innate advantages of the DARTS method, it is expected to be a powerful tool to accelerate SM target discovery, especially for bioactive natural products with unknown mechanisms.

GB-2 blocking the interaction between ACE2 and wild type and mutation of spike protein of SARS-CoV-2.

Since the start of the outbreak of coronavirus disease 2019 in Wuhan, China, there have been more than 150 million confirmed cases of the disease reported to the World Health Organization. The beta variant (B.1.351 lineage), the mutation lineages of SARS-CoV-2, had increase transmissibility and resistance to neutralizing antibodies due to multiple mutations in the spike protein. N501Y, K417N and E484K, in the receptor binding domain (RBD) region may induce a conformational change of the spike protein and subsequently increase the infectivity of the beta variant. The L452R mutation in the epsilon variant (the B.1.427/B.1.429 variants) also reduced neutralizing activity of monoclonal antibodies. In this study, we discovered that 300 µg/mL GB-2, from Tian Shang Sheng Mu of Chiayi Puzi Peitian Temple, can inhibit the binding between ACE2 and wild-type (Wuhan type) RBD spike protein. GB-2 can inhibit the binding between ACE2 and RBD with K417N-E484K-N501Y mutation in a dose-dependent manner. GB-2 inhibited the binding between ACE2 and the RBD with a single mutation (K417N or N501Y or L452R) except the E484K mutation. In the compositions of GB-2, glycyrrhiza uralensis Fisch. ex DC., theaflavin and (+)-catechin cannot inhibit the binding between ACE2 and wild-type RBD spike protein. Theaflavin 3-gallate can inhibit the binding between ACE2 and wild-type RBD spike protein. Our results suggest that GB-2 could be a potential candidate for the prophylaxis of some SARS-CoV-2 variants infection in the further clinical study because of its inhibition of binding between ACE2 and RBD with K417N-E484K-N501Y mutations or L452R mutation.

Mutual Interactions Between GnRH and Kisspeptin in GnRH- and Kiss-1-Expressing Immortalized Hypothalamic Cell Models.

Kisspeptin and gonadotropin-releasing hormone (GnRH) are central regulators of the hypothalamic-pituitary-gonadal axis and control female reproductive functions. Recently established mHypoA-50 and mHypoA-55 cells are immortalized hypothalamic neuronal cell models that originated from the anteroventral periventricular nucleus (AVPV) and arcuate nucleus (ARC) regions of the mouse hypothalamus, respectively. mHypoA-50 or mHypoA-55 cells were stimulated with kisspeptin-10 (KP10) and GnRH, after which the expression of kisspeptin and GnRH was determined. Primary cultures of fetal rat brain cells were also examined. mHypoA-50 and mHypoA-55 cells expressed mRNA for Kiss-1 (which encodes kisspeptin) and GnRH as well as receptors for kisspeptin and GnRH. We found that Kiss-1 mRNA expression was significantly increased in mHypoA-50 AVPV cells by KP10 and GnRH stimulation. Kisspeptin protein expression was also increased by KP10 and GnRH stimulation in these cells. In contrast, GnRH expression was unchanged in mHypoA-50 AVPV cells by KP10 and GnRH stimulation. In mHypoA-55 ARC cells, kisspeptin expression was also significantly increased at the mRNA and protein levels by KP10 and GnRH stimulation; however, GnRH expression was also upregulated by KP10 and GnRH stimulation in these cells. KP10 and estradiol (E2) both increased Kiss-1 gene expression in mHypoA-50 AVPV cells, but combined stimulation with KP10 and E2 did not potentiate their individual effects on Kiss-1 gene expression. On the other hand, E2 did not increase Kiss-1 gene expression in mHypoA-55 ARC cells, and the KP10-induced increase of Kiss-1 gene expression was inhibited in the presence of E2 in these cells. KP10 and GnRH significantly increased c-Fos protein expression in the mHypoA-50 AVPV and mHypoA-55 ARC cell lines. In primary cultures of fetal rat neuronal cells, KP10 significantly increased Kiss-1 gene expression, whereas GnRH significantly increased GnRH gene expression. We found that kisspeptin and GnRH affected Kiss-1- and GnRH-expressing hypothalamic cells and modulated Kiss-1 and/or GnRH gene expression with a concomitant increase in c-Fos protein expression. A mutual- or self-regulatory system might be present in Kiss-1 and/or GnRH neurons in the hypothalamus.

Insight into Functional Membrane Proteins by Solution NMR: The Human Bcl-2 Protein-A Promising Cancer Drug Target.

Evasion from programmed cell death (apoptosis) is the main hallmark of cancer and a major cause of resistance to therapy. Many tumors simply ensure survival by over-expressing the cell-protecting (anti-apoptotic) Bcl-2 membrane protein involved in apoptotic regulation. However, the molecular mechanism by which Bcl-2 protein in its mitochondrial outer membrane location protects cells remains elusive due to the absence of structural insight; and current strategies to therapeutically interfere with these Bcl-2 sensitive cancers are limited. Here, we present an NMR-based approach to enable structural insight into Bcl-2 function; an approach also ideal as a fragment-based drug discovery platform for further identification and development of promising molecular Bcl-2 inhibitors. By using solution NMR spectroscopy on fully functional intact human Bcl-2 protein in a membrane-mimicking micellar environment, and constructs with specific functions remaining, we present a strategy for structure determination and specific drug screening of functional subunits of the Bcl-2 protein as targets. Using 19F NMR and a specific fragment library (Bionet) with fluorinated compounds we can successfully identify various binders and validate our strategy in the hunt for novel Bcl-2 selective cancer drug strategies to treat currently incurable Bcl-2 sensitive tumors.

In silico Nigellidine (N. sativa) bind to viral spike/active-sites of ACE1/2, AT1/2 to prevent COVID-19 induced vaso-tumult/vascular-damage/comorbidity.

COVID-19, a global-pandemic binds human-lung-ACE2. ACE2 causes vasodilatation. ACE2 works in balance with ACE1. The vaso-status maintains blood-pressure/vascular-health which is demolished in Covid-19 manifesting aldosterone/salt-deregulations/inflammations/endothelial-dysfunctions/hyper-hypo- tension, sepsis/hypovolemic-shock and vessel-thrombosis/coagulations. Here, nigellidine, an indazole-alkaloid was analyzed by molecular-docking for binding to different Angiotensin-binding-proteins (enzymes, ACE1(6en5)/ACE2(4aph)/receptors, AT1(6os1)/AT2(5xjm)) and COVID-19 spike-glycoprotein(6vsb). Nigellidine strongly binds to the spike-protein at the hinge-region/active-site-opening which may hamper proper-binding of nCoV2-ACE2 surface. Nigellidine effectively binds in the Angiotensin- II binding-site/entry-pocket (-7.54 kcal/mol, -211.76, Atomic-Contact-Energy; ACE-value) of ACE2 (Ki 8.68 and 8.3 µmol) in comparison to known-binder EGCG (-4.53) and Theaflavin-di-gallate (-2.85). Nigellidine showed strong-binding (Ki, 50.93 µmol/binding-energy -5.48 kcal/mol) to mono/multi-meric ACE1. Moreover, it binds Angiotensin-receptors, AT1/AT2 (Ki, 42.79/14.22 µmol, binding-energy, -5.96/-6.61 kcal/mol) at active-sites, respectively. This article reports the novel binding of nigellidine and subsequent blockage of angiotensin-binding proteins. The ACEs-blocking could restore Angiotensin-level, restrict vaso-turbulence in Covid patients and receptor-blocking might stop inflammatory/vascular impairment. Nigellidine may slowdown the vaso-fluctuations due to Angiotensin-deregulations in Covid patients. Angiotensin II-ACE2 binding (ACE-value -294.81) is more favorable than nigellidine-ACE2. Conversely, nigellidine-ACE1 binding-energy/Ki is lower than nigellidine-ACE2 values indicating a balanced-state between constriction-dilatation. Moreover, nigellidine binds to the viral-spike, closer-proximity to its ACE2 binding-domain. Taken together, Covid patients/elderly-patients, comorbid-patients (with hypertensive/diabetic/cardiac/renal-impairment, counting >80% of non-survivors) could be greatly benefited.

Interaction of silymarin components and their sulfate metabolites with human serum albumin and cytochrome P450 (2C9, 2C19, 2D6, and 3A4) enzymes.

Silymarin is a mixture of flavonolignans isolated from the fruit of milk thistle (Silybum marianum (L.) Gaertner). Milk thistle extract is the active ingredient of several medications and dietary supplements to treat liver injury/diseases. After the oral administration, flavonolignans are extensively biotransformed, resulting in the formation of sulfate and/or glucuronide metabolites. Previous studies demonstrated that silymarin components form stable complexes with serum albumin and can inhibit certain cytochrome P450 (CYP) enzymes. Nevertheless, in most of these investigations, silybin was tested; while no or only limited information is available regarding other silymarin components and metabolites. In this study, the interactions of five silymarin components (silybin A, silybin B, isosilybin A, silychristin, and 2,3-dehydrosilychristin) and their sulfate metabolites were examined with human serum albumin and CYP (2C9, 2C19, 2D6, and 3A4) enzymes. Our results demonstrate that each compound tested forms stable complexes with albumin, and certain silymarin components/metabolites can inhibit CYP enzymes. Most of the sulfate conjugates were less potent inhibitors of CYP enzymes, but 2,3-dehydrosilychristin-19-O-sulfate showed the strongest inhibitory effect on CYP3A4. Based on these observations, the simultaneous administration of high dose silymarin with medications should be carefully considered, because milk thistle flavonolignans and/or their sulfate metabolites may interfere with drug therapy.

Pharmacokinetic / pharmacodynamic relationships of liposomal amphotericin B and miltefosine in experimental visceral leishmaniasis.

BACKGROUND: There is a continued need to develop effective and safe treatments for visceral leishmaniasis (VL). Preclinical studies on pharmacokinetics and pharmacodynamics of anti-infective agents, such as anti-bacterials and anti-fungals, have provided valuable information in the development and dosing of these agents. The aim of this study was to characterise the pharmacokinetic and pharmacodynamic properties of the anti-leishmanial drugs AmBisome and miltefosine in a preclinical disease model of VL. METHODOLOGY / PRINCIPAL FINDINGS: BALB/c mice were infected with L. donovani (MHOM/ET/67/HU3) amastigotes. Groups of mice were treated with miltefosine (orally, multi-dose regimen) or AmBisome (intravenously, single dose regimen) or left untreated as control groups. At set time points groups of mice were killed and plasma, livers and spleens harvested. For pharmacodynamics the hepatic parasite burden was determined microscopically from tissue impression smears. For pharmacokinetics drug concentrations were measured in plasma and whole tissue homogenates by LC-MS. Unbound drug concentrations were determined by rapid equilibrium dialysis. Doses exerting maximum anti-leishmanial effects were 40 mg/kg for AmBisome and 150 mg/kg (cumulatively) for miltefosine. AmBisome displayed a wider therapeutic range than miltefosine. Dose fractionation at a total dose of 2.5 mg/kg pointed towards concentration-dependent anti-leishmanial activity of AmBisome, favouring the administration of large doses infrequently. Protein binding was >99% for miltefosine and amphotericin B in plasma and tissue homogenates. CONCLUSION / SIGNIFICANCE: Using a PK/PD approach we propose optimal dosing strategies for AmBisome. Additionally, we describe pharmacokinetic and pharmacodynamic properties of miltefosine and compare our findings in a preclinical disease model to available knowledge from studies in humans. This approach also presents a strategy for improved use of animal models in the drug development process for VL.

Uranium directly interacts with the DNA repair protein poly (ADP-ribose) polymerase 1.

People living in southwest part of United States are exposed to uranium (U) through drinking water, air, and soil. U is radioactive, but independent of this radioactivity also has important toxicological considerations as an environmental metal. At environmentally relevant concentrations, U is both mutagenic and carcinogenic. Emerging evidence shows that U inhibits DNA repair activity, but how U interacts with DNA repair proteins is still largely unknown. Herein, we report that U directly interacts with the DNA repair protein, Protein Poly (ADP-ribose) Polymerase 1 (PARP-1) through direct binding with the zinc finger motif, resulting in zinc release from zinc finger and DNA binding activity loss of the protein. At the peptide level, instead of direct competition with zinc ion in the zinc finger motif, U does not show thermodynamic advantages over zinc. Furthermore, zinc pre-occupied PARP-1 zinc finger is insensitive to U treatment, but U bound to PARP-1 zinc finger can be partially replaced by zinc. These results provide mechanistic basis on molecular level to U inhibition of DNA repair.

Mechanism of self/nonself-discrimination in Brassica self-incompatibility.

Self-incompatibility (SI) is a breeding system that promotes cross-fertilization. In Brassica, pollen rejection is induced by a haplotype-specific interaction between pistil determinant SRK (S receptor kinase) and pollen determinant SP11 (S-locus Protein 11, also named SCR) from the S-locus. Although the structure of the B. rapa S9-SRK ectodomain (eSRK) and S9-SP11 complex has been determined, it remains unclear how SRK discriminates self- and nonself-SP11. Here, we uncover the detailed mechanism of self/nonself-discrimination in Brassica SI by determining the S8-eSRK-S8-SP11 crystal structure and performing molecular dynamics (MD) simulations. Comprehensive binding analysis of eSRK and SP11 structures reveals that the binding free energies are most stable for cognate eSRK-SP11 combinations. Residue-based contribution analysis suggests that the modes of eSRK-SP11 interactions differ between intra- and inter-subgroup (a group of phylogenetically neighboring haplotypes) combinations. Our data establish a model of self/nonself-discrimination in Brassica SI.

The mechanism of binding with the α-glucosidase in vitro and the evaluation on hypoglycemic effect in vivo: Cocrystals involving synergism of gallic acid and conformer.

Cocrystallization of Active Pharmaceutical Ingredients (API) with formers can induce positive or negative synergistic effects on activity; however, the underlying mechanism is unclear. In this study, we screened two cocrystals of gallic acid (GA): GA-p-aminobenzoic acid (cocrystal A) and GA-amino acetic acid (cocrystal B). Solubility, dissolution rate, and oral bioavailability and hypoglycemic effect of the two cocrystals were evaluated. Additionally, we examined the effect induced by cocrystallization of GA with each former on inhibition activity on α-glucosidase, a protein target involved in hypoglycemic effects. Cocrystals A and B were constructed in a 1:1 API/former molar ratio by CO⋯HN and OH⋯OC hydrogen bonds, respectively. As predicted, cocrystallization improved oral bioavailability; AUC0-∞s of cocrystal A and B were 2.24-fold and 1.70-fold higher than that of GA. Interestingly, the α-glucosidase inhibition rate increased with cocrystal A (i.e., positive synergism) and decreased with cocrystal B (i.e., negative synergism) compared to GA alone. For each cocrystal system, an obvious difference in the α-glucosidase inhibition rate between cocrystal and its physical mixture (PM) of API and former was observed. The 1H NMR analysis of two cocrystals and their respective PM indicated that hydrogen bond interactions between API and former molecules were just present in the solutions of cocrystal; but not in that of PMs. Molecular docking indicated that the hydrogen bonds between GA and CCF achieved binding with α-glucosidase in the form of supramolecular. Due to improvements in both oral bioavailability and α-glucosidase inhibition rate, the maximum hypoglycemic rate in diabetic mice treated with cocrystal A was 3.4-fold higher than that of GA alone. Conversely, although cocrystal B displayed improved bioavailability compared with GA alone, the maximum hypoglycemic rate remained almost unchanged due to the negative synergism on α-glucosidase inhibition activity of GA and amino acetic acid. Cocrystallization with each former induced variation not only on physiochemical properties and bioavailability but also on biological profiles involving inhibition rate on target proteins, which likely contributed to the observed positive and negative synergistic effects on API activity.

Role of carboxylic group pattern on protein surface in the recognition of iron oxide nanoparticles: A key for protein corona formation.

The knowledge of protein-nanoparticle interplay is of crucial importance to predict the fate of nanomaterials in biological environments. Indeed, protein corona on nanomaterials is responsible for the physiological response of the organism, influencing cell processes, from transport to accumulation and toxicity. Herein, a comparison using four different proteins reveals the existence of patterned regions of carboxylic groups acting as recognition sites for naked iron oxide nanoparticles. Readily interacting proteins display a distinctive surface distribution of carboxylic groups, recalling the geometric shape of an ellipse. This is morphologically complementary to nanoparticles curvature and compatible with the topography of exposed FeIII sites laying on the nanomaterial surface. The recognition site, absent in non-interacting proteins, promotes the nanoparticle harboring and allows the formation of functional protein coronas. The present work envisages the possibility of predicting the composition and the biological properties of protein corona on metal oxide nanoparticles.

Identification of novel CDK 9 inhibitors based on virtual screening, molecular dynamics simulation, and biological evaluation.

AIMS: Cyclin-dependent kinase 9 (CDK9) is a member of the CDK subfamily and plays a major role in the regulation of transcriptional elongation. It has attracted widespread attention as a therapeutic target for cancer. Here, we aimed to explore novel CDK 9 inhibitors by using a hybrid virtual screening strategy. MAIN METHODS: A hybrid virtual screening strategy was constructed with computer-aided drug design (CADD). First, compounds were filtered in accordance with Lipinski's rule of five and adsorption, distribution, metabolism, excretion, and toxicity (ADMET) properties. Second, a 3D-QSAR pharmacophore model was built and used as a 3D query to screen the obtained hit compounds. Third, the hit compounds were subjected to molecular docking studies. Fourth, molecular dynamics (MD) simulations were performed on CDK9 in complex with the final hits to examine the structural stability. Finally, CDK9 kinase biochemical assay was performed to identify the biological activity of the hit compounds. KEY FINDINGS: Seven hit compounds were screened out. These hit compounds showed drug-like properties in accordance with Lipinski's rule of five and ADMET. Complexes involving the six hit compounds bound to CDK9 exhibited good structural stability in the MD simulation. Furthermore, these six hit compounds had strong inhibitory activity against CDK9 kinase. In particular, hit 3 showed the most promising activity with the percentage of 71%. SIGNIFICANCE: The six hit compounds may be promising novel CDK9 inhibitors, and the hybrid virtual screening strategy designed in this study provides an important reference for the design and synthesis of novel CDK9 inhibitors.

Novel Compounds Identified by Structure-Based Prion Disease Drug Discovery Using In Silico Screening Delay the Progression of an Illness in Prion-Infected Mice.

The accumulation of abnormal prion protein (PrPSc) produced by the structure conversion of PrP (PrPC) in the brain induces prion disease. Although the conversion process of the protein is still not fully elucidated, it has been known that the intramolecular chemical bridging in the most fragile pocket of PrP, known as the "hot spot," stabilizes the structure of PrPC and inhibits the conversion process. Using our original structure-based drug discovery algorithm, we identified the low molecular weight compounds that predicted binding to the hot spot. NPR-130 and NPR-162 strongly bound to recombinant PrP in vitro, and fragment molecular orbital (FMO) analysis indicated that the high affinity of those candidates to the PrP is largely dependent on nonpolar interactions, such as van der Waals interactions. Those NPRs showed not only significant reduction of the PrPSc levels but also remarkable decrease of the number of aggresomes in persistently prion-infected cells. Intriguingly, treatment with those candidate compounds significantly prolonged the survival period of prion-infected mice and suppressed prion disease-specific pathological damage, such as vacuole degeneration, PrPSc accumulation, microgliosis, and astrogliosis in the brain, suggesting their possible clinical use. Our results indicate that in silico drug discovery using NUDE/DEGIMA may be widely useful to identify candidate compounds that effectively stabilize the protein.

Synthesis and Biochemical Evaluation of Noncyclic Nucleotide Exchange Proteins Directly Activated by cAMP 1 (EPAC1) Regulators.

Exchange proteins directly activated by cAMP (EPAC) play a central role in various biological functions, and activation of the EPAC1 protein has shown potential benefits for the treatment of various human diseases. Herein, we report the synthesis and biochemical evaluation of a series of noncyclic nucleotide EPAC1 activators. Several potent EPAC1 binders were identified including 25g, 25q, 25n, 25u, 25e, and 25f, which promote EPAC1 guanine nucleotide exchange factor activity in vitro. These agonists can also activate EPAC1 protein in cells, where they exhibit excellent selectivity toward EPAC over protein kinase A and G protein-coupled receptors. Moreover, 25e, 25f, 25n, and 25u exhibited improved selectivity toward activation of EPAC1 over EPAC2 in cells. Of these, 25u was found to robustly inhibit IL-6-activated signal transducer and activator of transcription 3 (STAT3) and subsequent induction of the pro-inflammatory vascular cell adhesion molecule 1 (VCAM1) cell-adhesion protein. These novel EPAC1 activators may therefore act as useful pharmacological tools for elucidation of EPAC function and promising drug leads for the treatment of relevant human diseases.

Pharmacological Characterization of a Betaine/GABA Transporter 1 (BGT1) Inhibitor Displaying an Unusual Biphasic Inhibition Profile and Anti-seizure Effects.

Focal epileptic seizures can in some patients be managed by inhibiting γ-aminobutyric acid (GABA) uptake via the GABA transporter 1 (GAT1) using tiagabine (Gabitril®). Synergistic anti-seizure effects achieved by inhibition of both GAT1 and the betaine/GABA transporter (BGT1) by tiagabine and EF1502, compared to tiagabine alone, suggest BGT1 as a target in epilepsy. Yet, selective BGT1 inhibitors are needed for validation of this hypothesis. In that search, a series of BGT1 inhibitors typified by (1R,2S)-2-((4,4-bis(3-methylthiophen-2-yl)but-3-en-yl)(methyl)amino)cyclohexanecarboxylic acid (SBV2-114) was developed. A thorough pharmacological characterization of SBV2-114 using a cell-based [3H]GABA uptake assay at heterologously expressed BGT1, revealed an elusive biphasic inhibition profile with two IC50 values (4.7 and 556 µM). The biphasic profile was common for this structural class of compounds, including EF1502, and was confirmed in the MDCK II cell line endogenously expressing BGT1. The possibility of two binding sites for SBV2-114 at BGT1 was assessed by computational docking studies and examined by mutational studies. These investigations confirmed that the conserved residue Q299 in BGT1 is involved in, but not solely responsible for the biphasic inhibition profile of SBV2-114. Animal studies revealed anti-seizure effects of SBV2-114 in two mouse models, supporting a function of BGT1 in epilepsy. However, as SBV2-114 is apparent to be rather non-selective for BGT1, the translational relevance of this observation is unknown. Nevertheless, SBV2-114 constitutes a valuable tool compound to study the molecular mechanism of an emerging biphasic profile of BGT1-mediated GABA transport and the putative involvement of two binding sites for this class of compounds.

Virtual screening of antitumour phytochemical against peroxisome proliferators activated receptor proteins PPARs.

Cancers are caused by the defects in apoptosis process which leads to uncontrolled proliferation, therefore, most attractive drug target discovery strategy is to find ligands which have the ability to activate or regulate the apoptotic machinery. Peroxisome-proliferator-activated receptors (PPARs) are nuclear hormone receptors their over expression is observed in many tumours and contributes to chemotherapy resistance. The goal of this study to scrutinized antitumor phytochemicals from Alysicarpus bupleurifolius, Piper nigrum and Plumeria obtuse and potential energy values render from interactions between active site residues and ligands. The potential phytochemicals with significant binding affinity are ursolic acid, cis-4-decenoic acid and p-coumaric acid respectively most effective compounds in high throughput virtual screening belongs to Plumeria obtuse against PPARs associated with tumour development and progression. This modern drug designing modeling in silico approach, therefore, identifies the potential leads against over expressed tumours.