A Diversifiable Synthetic Platform for the Discovery of New Carbasugar SGLT2 Inhibitors Using Azide-Alkyne Click Chemistry.

Sodium-glucose cotransporter 2 (SGLT2) inhibitors are clinically available to control blood glucose levels in diabetic patients via an insulin-independent mechanism. It was found that some carbasugar analogs of known SGLT2 inhibitors exert a high inhibiting ability toward SGLT2 and have a prolonged blood glucose lowering effect. In this study, we designed new candidates of carbasugar SGLT2 inhibitor that can be synthesized using copper-catalyzed azide-alkyne cycloaddition (CuAAC) into an aromatic ring, which is a part of the pharmacophore at the final stage in the synthetic protocol for the easier discovery of superior SGLT2 inhibitors. Based on the results of molecular docking studies, some selected compounds have been synthesized. Evaluation of these compounds using a cell-based assay revealed that the majority of these compounds had SGLT2 inhibitory activity in a dose-dependent manner. The SGLT2 inhibitory activity of 7b and 7c was almost equal to that of SGLT2 inhibitors in current use. Furthermore, molecular dynamics simulations also revealed that 7c is a promising novel SGLT2 inhibitor.

Antiviral drug discovery by targeting the SARS-CoV-2 polyprotein processing by inhibition of the main protease.

The spread of SARS-CoV-2, the causative agent for COVID-19, has led to a global and deadly pandemic. To date, few drugs have been approved for treating SARS-CoV-2 infections. In this study, a structure-based approach was adopted using the SARS-CoV-2 main protease (Mpro) and a carefully selected dataset of 37,060 compounds comprising Mpro and antiviral protein-specific libraries. The compounds passed two-step docking filtration, starting with standard precision (SP) followed by extra precision (XP) runs. Fourteen compounds with the highest XP docking scores were examined by 20 ns molecular dynamics simulations (MDs). Based on backbone route mean square deviations (RMSD) and molecular mechanics/generalized Born surface area (MM/GBSA) binding energy, four drugs were selected for comprehensive MDs analysis at 100 ns. Results indicated that birinapant, atazanavir, and ritonavir potently bound and stabilized SARS-CoV-2 Mpro structure. Binding energies higher than -102 kcal/mol, RMSD values <0.22 nm, formation of several hydrogen bonds with Mpro, favourable electrostatic contributions, and low radii of gyration were among the estimated factors contributing to the strength of the binding of these three compounds with Mpro. The top two compounds, atazanavir and birinapant, were tested for their ability to prevent SARS-CoV-2 plaque formation. At 10 µM of birinapant concentration, antiviral tests against SARS-CoV-2 demonstrated a 37% reduction of virus multiplication. Antiviral assays demonstrated that birinapant has high anti-SARS-CoV-2 activity in the low micromolar range, with an IC50 value of 18 ± 3.6 µM. Therefore, birinapant is a candidate for further investigation to determine whether it is a feasible therapy option.

The emerging SARS-CoV-2 papain-like protease: Its relationship with recent coronavirus epidemics.

The papain-like protease (PLpro ) is an important enzyme for coronavirus polyprotein processing, as well as for virus-host immune suppression. Previous studies reveal that a molecular analysis of PLpro indicates the catalytic activity of viral PLpro and its interactions with ubiquitin. By using sequence comparisons, molecular models, and protein-protein interaction maps, PLpro was compared in the three recorded fatal CoV epidemics, which involved severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), severe acute respiratory syndrome CoV (SARS-CoV), and Middle East respiratory syndrome coronavirus (MERS-CoV). The pairwise sequence comparison of SARS-CoV-2 PLpro indicated similarity percentages of 82.59% and 30.06% with SARS-CoV PLpro and MERS-CoV PLpro , respectively. In comparison with SARS-CoV PLpro , in SARS-CoV-2, the PLpro had a conserved catalytic triad of C111, H278, and D293, with a slightly lower number of polar interface residues and of hydrogen bonds, a higher number of buried interface sizes, and a lower number of residues that interact with ubiquitin and PLpro . These features might contribute to a similar or slightly lower level of deubiquitinating activity in SARS-CoV-2 PLpro. It was, however, a much higher level compared to MERS-CoV, which contained amino acid mutations and a low number of polar interfaces. SARS-CoV-2 PLpro and SARS-CoV PLpro showed almost the same catalytic site profiles, interface area compositions and polarities, suggesting a general similarity in deubiquitination activity. Compared with MERS-CoV, SARS-CoV-2 had a higher potential for binding interactions with ubiquitin. These estimated parameters contribute to the knowledge gap in understanding how the new virus interacts with the immune system.

Repurposing FDA-approved phytomedicines, natural products, antivirals and cell protectives against SARS-CoV-2 (COVID-19) RNA-dependent RNA polymerase.

Following the recent emergence of SARS-CoV-2 or coronavirus disease 2019 (COVID-19), drug discovery and vaccine design to combat this fatal infection are critical. In this study, an essential enzyme in the SARS-CoV-2 replication machinery, RNA-dependent RNA polymerase (RDRP), is targeted in a virtual screening assay using a set of 1,664 FDA-approved drugs, including sets of botanical and synthetic derivatives. A set of 22 drugs showed a high docking score of >-7. Notably, approximately one-third of the top hits were either from natural products or biological molecules. The FDA-approved phytochemicals were sennosides, digoxin, asiaticoside, glycyrrhizin, neohesperidin, taxifolin, quercetin and aloin. These approved natural products and phytochemicals are used as general tonics, antioxidants, cell protectives, and immune stimulants (nadid, thymopentin, asiaticoside, glycyrrhizin) and in other miscellaneous systemic or topical applications. A comprehensive analysis was conducted on standard precision and extra precision docking, two-step molecular dynamics simulations, binding energy calculations and a post dynamics analysis. The results reveal that two drugs, docetaxel and neohesperidin, showed strong binding profiles with SARS CoV-2 RdRP. These results can be used as a primer for further drug discovery studies in the treatment of COVID-19. This initiative repurposes safe FDA-approved drugs against COVID-19 RdRP, providing a rapid channel for the discovery and application of new anti-CoV therapeutics.

Sulfonamide antibiotics inhibit RNAi by binding to human Argonaute protein 2 PAZ.

We found that sulfisomidine, a sulfonamide antibiotic, potently binds to the Piwi/Argonaute/Zwille (PAZ) domain of human Argonaute protein 2 and inhibits RNA interference (RNAi). To elucidate the effect on RNAi of strong affinity of the 3'-ends in small interfering RNA (siRNA) to the PAZ domain, chemically modified siRNAs bearing sulfisomidine at the 3'-end were synthesized.

A pilot study on the pharmacokinetics of a single intramuscular injection of cefquinome in Arabian camel calves.

This study was conducted to evaluate the pharmacokinetics of cefquinome in camel calves after a single intramuscular injection in a dose of 2 mg/kg body weight (kg b. w.). Cefquinome concentrations were measured by ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS/MS). A non-compartmental pharmacokinetic model was used to fit the time-concentration curve and estimate the pharmacokinetic parameters. The peak serum concentration (Cmax) was 28.4 µg/mL at the time of maximum concentration (Tmax) of 25 min. The elimination half-life (t1/2) was 17.4 h. The area under the concentration-time curve (AUC0-∞) was 103.7 µg/ml-1h and the mean residence time (MRT0-∞) was 21.3 h. In comparison with other animal species, the pharmacokinetics of cefquinome in Arabian camel calves showed faster absorption from the site of injection and slower elimination. Since cefquinome, as other beta-lactams, is a time-dependent antimicrobial agent, a single dose of 2 mg/kg b. w. might be sufficient against the most sensitive organisms in camel calves owing to its prolonged elimination phase. However, dose readjustment is required for cases needing concentrations above 2 µg/mL for 12 h or above 1 µg/mL for 24 h.

ABCE1 Acts as a Positive Regulator of Exogenous RNA Decay.

The 2'-5'-oligoadenylate synthetase (OAS)/RNase L system protects hosts against pathogenic viruses through cleavage of the exogenous single-stranded RNA. In this system, an evolutionally conserved RNA quality control factor Dom34 (known as Pelota (Pelo) in higher eukaryotes) forms a surveillance complex with RNase L to recognize and eliminate the exogenous RNA in a manner dependent on translation. Here, we newly identified that ATP-binding cassette sub-family E member 1 (ABCE1), which is also known as RNase L inhibitor (RLI), is involved in the regulation of exogenous RNA decay. ABCE1 directly binds to form a complex with RNase L and accelerates RNase L dimer formation in the absence of 2'-5' oligoadenylates (2-5A). Depletion of ABCE1 represses 2-5A-induced RNase L activation and stabilizes exogenous RNA to a level comparable to that seen in RNase L depletion. The increased half-life of the RNA by the single depletion of either protein is not significantly affected by the double depletion of both proteins, suggesting that RNase L and ABCE1 act together to eliminate exogenous RNA. Our results indicate that ABCE1 functions as a positive regulator of exogenous RNA decay rather than an inhibitor of RNase L.

Practical and reliable synthesis of 2',3',5',5″-tetradeuterated uridine derivatives.

Deuterated drugs are valuable in the fields of drug discovery and medicinal chemistry. 2',3',5',5″-tetradeuterated uridine derivatives were synthesised from 2,3,5,5'-selectively tetradeuterated ribose using Sajiki's H-D exchanged Ru/C-H2-D2O-NaOH system and silyl-Hilbert-Johnson methods. The total deuterium content of the tetradeuterated uridines was over 92% using either basic or acidic reaction conditions. These derivatives would be expected as building blocks for the synthesis of deuterium-substituted nucleic acid probes for tracking the pharmacokinetics of nucleic acid drugs.

Synthetic microRNA-205 exhibited tumour suppression in spontaneous canine malignant melanoma by intratumoral injection.

MicroRNAs (miRNA) are small, noncoding RNA molecules consisting of 18 to 25 nucleotides. Malignant melanomas (MMs) are one of the most common malignancies in both dogs and humans. We previously reported that chemically modified synthetic miRNA-205 (miR-205BP/S3) inhibits melanoma growth in vitro and in vivo. The present study aimed to evaluate the efficacy of intratumoral administration of synthetic miR-205 for spontaneous CMMs and to evaluate its potential as systemic therapy. Ten dogs with various stages of MM were treated with miR-205BP/S3 injected into tumours. Adverse effects (AEs) were assessed in accordance with the Veterinary Cooperative Oncology Group-Common Terminology Criteria for Adverse Events (VCOG-CTCAE) v1.1 guidelines. Five cases attained complete remission (CR), three attained stable disease (SD), and two cases displayed characteristics of progressive disease (PD). In all cases, no changes were observed in the blood parameters upon miRNA administration, and miR-205BP/S3 administration did not yield any side effects. The present results suggest that intratumoral administration of miR-205BP/S3 is a potentially applicable treatment for canine melanoma.

The structural basis of unique substrate recognition by Plasmodium thymidylate kinase: Molecular dynamics simulation and inhibitory studies.

Plasmodium falciparum thymidylate kinase (PfTMK) showed structural and catalytic distinctions from the host enzyme rendering it a hopeful antiprotozoal drug target. Despite the comprehensive enzymologic, structural, inhibitory and chemical synthesis approaches targeting this enzyme, the elucidation of the exact mechanism underlying the recognition of the atypical purine substrates remains to be determined. In this study, molecular dynamics (MD) simulation of a broad range of substrates and inhibitors as well as the inhibitory properties of deoxyguanosine (dG) derivatives were used to assess the PfTMK substructure molecular rearrangements. The estimated changes during the favourable binding of high affinity substrate (TMP) include lower interaction with P-loop, free residue fluctuations of the lid domain and the average RMSD value. The RMSD of TMP complex was higher and more rapidly stabilized than the dGMP complex. The lid domain flexibility is severely affected by dGMP and ß-thymidine derivatives, while being partially fluctuating with other thymidine derivatives. The TMK-purine (dGMP) complex was slowly and gradually stabilized with lower over all structure flexibility and residue fluctuations especially at the lid domain, which closes the active site during its catalytic state. Thymidine derivatives allow structure flexibility of the lid domain being highly fluctuating in α- and ß-thymidine derivatives and TMP. dG derivatives remains less efficient than thymidine derivatives in inhibiting TMK. The variations in the structural dynamics of the P-loop and lid domain in response to TMP or dGMP might favour thymidine-based compounds. The provided MD simulation strategy can be used for predicating structural changes in PfTMK during lead optimization.

Dom34 mediates targeting of exogenous RNA in the antiviral OAS/RNase L pathway.

The 2'-5'-oligoadenylate synthetase (OAS)/RNase L pathway is an innate immune system that protects hosts against pathogenic viruses and bacteria through cleavage of exogenous single-stranded RNA; however, this system's selective targeting mechanism remains unclear. Here, we identified an mRNA quality control factor Dom34 as a novel restriction factor for a positive-sense single-stranded RNA virus. Downregulation of Dom34 and RNase L increases viral replication, as well as half-life of the viral RNA. Dom34 directly binds RNase L to form a surveillance complex to recognize and eliminate the exogenous RNA in a manner dependent on translation. Interestingly, the feature detected by the surveillance complex is not the specific sequence of the viral RNA but the 'exogenous nature' of the RNA. We propose the following model for the selective targeting of exogenous RNA; OAS3 activated by the exogenous RNA releases 2'-5'-oligoadenylates (2-5A), which in turn converts latent RNase L to an active dimer. This accelerates formation of the Dom34-RNase L surveillance complex, and its selective localization to the ribosome on the exogenous RNA, thereby promoting degradation of the RNA. Our findings reveal that the selective targeting of exogenous RNA in antiviral defense occurs via a mechanism similar to that in the degradation of aberrant transcripts in RNA quality control.

Synthesis of cationic glucosamino nucleic acids for stabilizing oligonucleotides.

Glucosamino nucleic acids (GANAs) bearing a ß-N-glycoside bond between carbon 1 of the glucosamine and the nucleobase nitrogen were synthesized and incorporated into oligonucleotides (4',6'-GANA and 3',6'-GANA). The thermal stability of oligonucleotide duplexes containing the GANA zwitterionic nucleotides was also investigated.

Impairment of K-Ras signaling networks and increased efficacy of epidermal growth factor receptor inhibitors by a novel synthetic miR-143.

Despite considerable research on K-Ras inhibitors, none had been established until now. We synthesized nuclease-resistant synthetic miR-143 (miR-143#12), which strongly silenced K-Ras, its effector signal molecules AKT and ERK, and the K-Ras activator Sos1. We examined the anti-proliferative effect of miR-143#12 and the mechanism in human colon cancer DLD-1 cell (G13D) and other cell types harboring K-Ras mutations. Cell growth was markedly suppressed in a concentration-dependent manner by miR-143#12 (IC50 : 1.32 nmol L-1 ) with a decrease in the K-Ras mRNA level. Interestingly, this mRNA level was also downregulated by either a PI3K/AKT or MEK inhibitor, which indicates a positive circuit of K-Ras mRNA expression. MiR-143#12 silenced cytoplasmic K-Ras mRNA expression and impaired the positive circuit by directly targeting AKT and ERK mRNA. Combination treatment with miR-143#12 and a low-dose EGFR inhibitor induced a synergistic inhibition of growth with a marked inactivation of both PI3K/AKT and MAPK/ERK signaling pathways. However, silencing K-Ras by siR-KRas instead of miR-143#12 did not induce this synergism through the combined treatment with the EGFR inhibitor. Thus, miR-143#12 perturbed the K-Ras expression system and K-Ras activation by silencing Sos1 and, resultantly, restored the efficacy of the EGFR inhibitors. The in vivo results also supported those of the in vitro experiments. The extremely potent miR-143#12 enabled us to understand K-Ras signaling networks and shut them down by combination treatment with this miRNA and EGFR inhibitor in K-Ras-driven colon cancer cell lines.

Reduction-Responsive DNA Duplex Containing O 6-Nitrobenzyl-Guanine.

Stimuli-controlled structural transitions of nucleic acids have received growing attentions owing to their potential applications in the fields of chemical and synthetic biology. Here, we describe the development of reduction-responsive deoxyribonucleic acid (DNA) duplexes, in which guanine rings bearing a reduction-responsive cleavable nitrobenzyl (NB) group at the O 6 position (GNB) are introduced at defined positions. We demonstrate that the artificial NB group can be removed in response to reduction stimulus without the dissociation of the intermolecular duplex structure, which comprises a G-quadruplex forming nucleic acid strand with one GNB and its complementary sequence with one mismatch pair. Meanwhile, another duplex that comprised a G-quadruplex forming nucleic acid strand with two GNB and its complementary sequence with three mismatch pairs exhibited reduction-responsive structural transitions from intermolecular duplex to intramolecular quadruplex. These findings might be useful for the development of DNA architectures endowed with reduction-responsive functions.

Molecular dynamics and binding selectivity of nucleotides and polynucleotide substrates with EIF2C2/Ago2 PAZ domain.

RNA interference (RNAi) constitutes a major target in drug discovery. Recently, we reported that the Argonaute protein 2 (Ago2) PAZ domain selectively binds with all ribonucleotides except adenine and poorly recognizes deoxyribonucleotides. The binding properties of the PAZ domain with polynucleotides and the molecular mechanisms of substrates' selectivity remains unclear. In this study, the binding potencies of polynucleotides and the associated conformational and dynamic changes in PAZ domain are investigated. Coinciding with nucleotides' binding profile with the PAZ domain, polyuridylate (PolyU) and polycytidylate (PolyC) were potent binders. However, KdPolyU and KdPolyC were 15.8 and 9.3µM, respectively. In contrast, polyadenylate (PolyA) binding was not detectable. Molecular dynamics (MD) simulation revealed the highest change in root mean square deviation (RMSD) with ApoPAZ or PAZ domain bound with experimentally approved, low affinity substrates, whereas stronger binding substrates such as UMP or PolyU showed minimal RMSD changes. The loop between α3 and ß5 in the ß-hairpin subdomain showed the most responsive change in RMSD, being highly movable in the ApoPAZ and PAZ-AMP complex. Favorable substrate recognition was associate with moderate change in secondary structure content. In conclusion, the PAZ domain retains differential substrate selectivity associated with corresponding dynamic and structural changes upon binding.

Introduction of 2-O-benzyl abasic nucleosides to the 3'-overhang regions of siRNAs greatly improves nuclease resistance.

Chemically modified siRNAs containing 2-O-benzyl-1-deoxy-d-ribofuranose (RHOBn) in their 3'-overhang region were significantly more resistant towards serum nucleases than siRNAs possessing the natural nucleoside in this region. The knockdown efficacies and binding affinities of these modified siRNAs to the recombinant human Argonaute protein 2 (hAgo2) PAZ domain were comparable with that of siRNA with a thymidine dimer at the 3'-end.

Nucleobase azide-ethynylribose click chemistry contributes to stabilizing oligonucleotide duplexes and stem-loop structures.

The formation of 1,4-disubstituted 1,2,3-triazoles through copper-catalyzed azide-alkyne cycloaddition (CuAAC) in oligonucleotides bearing 1-deoxy-1-ethynyl-ß-d-ribofuranose (RE) can have a positive impact on the stability of oligonucleotide duplexes and stem-loop structures.

Epidemiological, molecular characterization and antibiotic resistance of Salmonella enterica serovars isolated from chicken farms in Egypt.

BACKGROUND: Salmonella is one of major causes of foodborne outbreaks globally. This study was conducted to estimate the prevalence, typing and antibiotic susceptibilities of Salmonella enterica serovars isolated from 41 broiler chicken farms located in Kafr El-Sheikh Province in Northern Egypt during 2014-2015. The clinical signs and mortalities were observed. RESULTS: In total 615 clinical samples were collected from broiler flocks from different organs (liver, intestinal content and gall bladder). Salmonella infection was identified in 17 (41%) broiler chicken flocks and 67 Salmonella isolates were collected. Recovered isolates were serotyped as 58 (86.6%) S. enterica serovar Typhimurium, 6 (9%) S. enterica serovar Enteritidis and 3 (4.5%) were non-typable. The significant high mortality rate was observed only in 1-week-old chicks. sopE gene was detected in 92.5% of the isolates which indicating their ability to infect humans. All S. enterica serovar Enteritidis isolates were susceptible to all tested antimicrobials. The phenotypically resistant S. enterica serovar Typhimurium isolates against ampicillin, tetracycline, sulphamethoxazole and chloramphenicol were harbouring BlaTEM, (tetA and tetC), (sul1 and sul3) and (cat1 and floR), respectively. The sensitivity rate of S. enterica serovar Typhimurium to gentamycin, trimethoprim/sulphamethoxazole and streptomycin were 100, 94.8, 89.7%, respectively. The silent streptomycin antimicrobial cassettes were detected in all Salmonella serovars. A class one integron (dfrA12, orfF and aadA2) was identified in three of S. enterica serovar Typhimurium strains. CONCLUSIONS: To the best of our knowledge, this study considered first report discussing the prevalence, genotyping, antibiotic susceptibility and public health significance of S. enterica serovars in broilers farms of different ages in Delta Egypt. Further studies are mandatory to verify the location of some resistance genes that are within or associated with the class one integron.

Four-and-a-half LIM Domains 1 (FHL1) Protein Interacts with the Rho Guanine Nucleotide Exchange Factor PLEKHG2/FLJ00018 and Regulates Cell Morphogenesis.

PLEKHG2/FLJ00018 is a Gßγ-dependent guanine nucleotide exchange factor for the small GTPases Rac and Cdc42 and has been shown to mediate the signaling pathways leading to actin cytoskeleton reorganization. Here we showed that the zinc finger domain-containing protein four-and-a-half LIM domains 1 (FHL1) acts as a novel interaction partner of PLEKHG2 by the yeast two-hybrid system. Among the isoforms of FHL1 (i.e. FHL1A, FHL1B, and FHL1C), FHL1A and FHL1B interacted with PLEKHG2. We found that there was an FHL1-binding region at amino acids 58-150 of PLEKHG2. The overexpression of FHL1A but not FHL1B enhanced the PLEKHG2-induced serum response element-dependent gene transcription. The co-expression of FHL1A and Gßγ synergistically enhanced the PLEKHG2-induced serum response element-dependent gene transcription. Increased transcription activity was decreased by FHL1A knock-out with the CRISPR/Cas9 system. Compared with PLEKHG2-expressing cells, the number and length of finger-like protrusions were increased in PLEKHG2-, Gßγ-, and FHL1A-expressing cells. Our results provide evidence that FHL1A interacts with PLEKHG2 and regulates cell morphological change through the activity of PLEKHG2.

Evolution of camel CYP2E1 and its associated power of binding toxic industrial chemicals and drugs.

Camels are raised in harsh desert environment for hundreds of years ago. By modernization of live and the growing industrial revolution in camels rearing areas, camels are exposed to considerable amount of chemicals, industrial waste, environmental pollutions and drugs. Furthermore, camels have unique gene evolution of some genes to withstand living in harsh environments. In this work, the camel cytochrome P450 2E1 (CYP2E1) is compromised to detect its evolution rate and its power to bind with various chemicals, protoxins, procarcinogens, industrial toxins and drugs. In comparison with human CYP2E1, camel CYP2E1 more efficiently binds to small toxins as aniline, benzene, catechol, amides, butadiene, toluene and acrylamide. Larger compounds were more preferentially bound to the human CYP2E1 in comparison with camel CYP2E1. The binding of inhalant anesthetics was almost similar in both camel and human CYP2E1 coinciding with similar anesthetic effect as well as toxicity profiles. Furthermore, evolutionary analysis indicated the high evolution rate of camel CYP2E1 in comparison with human, farm and companion animals. The evolution rate of camel CYP2E1 was among the highest evolution rate in a subset of 57 different organisms. These results indicate rapid evolution and potent toxin binding power of camel CYP2E1.