Identification of antiviral phytochemicals from cranberry as potential inhibitors of SARS-CoV-2 main protease (Mpro).

Cranberry phytochemicals are known to possess antiviral activities. In the current study, we explored the therapeutic potential of cranberry against SARS-CoV-2 by targeting its main protease (Mpro) enzyme. Firstly, phytochemicals of cranberry origin were identified from three independent databases. Subsequently, virtual screening, using molecular docking and molecular dynamics simulation approaches, led to the identification of three lead phytochemicals namely, cyanidin 3-O-galactoside, ß-carotene and epicatechin. Furthermore, in vitro enzymatic assays revealed that cyanidin 3-O-galactoside had the highest inhibitory potential with IC50 of 9.98 µM compared to the other two phytochemicals. Cyanidin 3-O-galactoside belongs to the class of anthocyanins. Anthocyanins extracted from frozen cranberry also exhibited the highest inhibitory potential with IC50 of 23.58 µg/ml compared to the extracts of carotenoids and flavanols, the class for ß-carotene and epicatechin, respectively. Finally, we confirm the presence of the phytochemicals in the cranberry extracts using targeted LC-MS/MS analysis. Our results, therefore, indicate that the identified cranberry-derived bioactive compounds as well as cranberry could be used for therapeutic interventions against SARS-CoV-2.

Repurposing drugs targeting metabolic diseases for cancer therapeutics.

Hurdles in the identification of new drugs for cancer treatment have made drug repurposing an increasingly appealing alternative. The approach involves the use of old drugs for new therapeutic purposes. It is cost-effective and facilitates rapid clinical translation. Given that cancer is also considered a metabolic disease, drugs for metabolic disorders are being actively repurposed for cancer therapeutics. In this review, we discuss the repurposing of such drugs approved for two major metabolic diseases, diabetes and cardiovascular disease (CVD), which have shown potential as anti-cancer treatment. We also highlight the current understanding of the cancer signaling pathways that these drugs target.

In silico and in vitro assays reveal potential inhibitors against 3CLpro main protease of SARS-CoV-2.

The COVID-19 pandemic, caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is not showing any sign of slowing down even after the ongoing efforts of vaccination. The threats of new strains are concerning, as some of them are more infectious than the original one. A therapeutic against the disease is, therefore, of urgent need. Here, we use the DrugBank database to screen for potential inhibitors against the 3CLpro main protease of SARS-CoV-2. Instead of using the traditional approach of computational screening by docking, we developed a kernel ridge regressor (using a part of the docking data) to predict the binding energy of ligands. We used this model to screen the DrugBank database and shortlist two lead candidates (bromocriptine and avoralstat) for in vitro enzymatic study. Our results show that the 3CLpro enzyme activity in presence of 100 µM concentration of bromocriptine and avoralstat is 9.9% and 15.9%, respectively. Remarkably, bromocriptine exhibited submicromolar IC50 of 130 nM (0.13 µM). Avoralstat showed an IC50 of 2.16 µM. Further, the interactions of both drugs with 3CLpro were analyzed using molecular dynamics simulations of 100 ns. Results indicate that both ligands are stable in the binding pocket of the 3CLpro receptor. In addition, the MM-PBSA analysis revealed that bromocriptine (-29.37 kcal/mol) has a lower binding free energy compared to avoralstat (-6.91 kcal/mol). Further, hydrogen bond analysis also showed that bromocriptine interacts with the two catalytic residues, His41 and Cys145, more frequently than avoralstat.Communicated by Ramaswamy H. Sarma.

Identification of Brugia malayi in dogs in Kerala, India.

Identification of filarial species in dogs is clinically important because of zoonotic concerns and therapeutic implications. The present study was carried out to identify the filarial parasites causing microfilaraemia in dogs in Thrissur District, Kerala- an endemic area for human Brugian filariasis. Out of the 1600 dogs screened by wet blood film examination, 130 were positive for microfilariasis. Giemsa staining of blood smears revealed that 90 out of 130 dogs had unsheathed microfilariae, 24 had sheathed microfilariae and 16 had combined infection of sheathed and unsheathed microfilariae. Results of micrometry and histochemical staining of the sheathed microfilariae were in conformity with that of Brugia malayi. The DNA isolated from the sheathed microfilariae amplified the primers specific for the Hha 1 repeats of the B. malayi. Cloning and sequencing revealed that the amplified fragment corresponded to the 140-292 base pairs of the 320 base pair Hha1 repeat of Brugia malayi. The amplified DNA fragment also contained restriction sites for Alu 1 and Rsa 1which confirmed that the present isolate is Brugia malayi. The present study confirmed the presence of B. malayi in dogs in Kerala, India.

Immunoblotting analysis of canine Brugia malayi microfilarial antigens.

Brugia malayi is a filarial parasite that causes lymphatic filariasis in human beings. Kerala (India) is endemic for human Brugian filariasis. B. malayi microfilariae, similar to that causing filariasis in human beings, have been identified in dogs in Kerala. This is the first report of western blotting analysis of canine B. malayi microfilarial proteins. SDS PAGE analysis of B. malayi microfilarial protein revealed 5 major protein bands with molecular weights of 125, 80, 64, 54 and 27 kDa. Among these, the prominent bands were those having molecular weights of 64, 54 and 27 kDa. We raised polyclonal antibodies against these somatic proteins of dog microfilariae in a rabbit. The polyclonal antibodies recognized predominantly the 54 kDa and 64 kDa antigens in a Western blot analysis. Based on previous publications with B. malayi, these two protein bands appear to be important for diagnosis and for vaccine development against lymphatic filariasis.

Identification of Dermatophilus congolensis from lower leg dermatitis of cattle in Kerala, India.

This study was conducted to identify the aetiological agents associated with a particular type of lower leg dermatitis, locally called pododermatitis, among dairy cattle in Kerala. Skin scabs and scrapings were collected aseptically from 82 naturally occurring cases of lower leg dermatitis in cattle and were subjected to direct microscopical examination and bacterial and fungal culture. Microscopical examination of the skin scrapings with 10% potassium hydroxide revealed fungal spores in hair shafts from only two samples and did not reveal the presence of mites or other parasites. Fungal culture yielded dermatophytes from only five samples; these were identified as Trichophyton mentagrophytes in two cases, T verrucosum in one case, Epidermophyton floccosum in one case and Microsporum nanum in one case. Microscopical examination of Giemsa- and Gram-stained smears of the scab material from the lesions from 72 cases revealed characteristic Gram-positive septate branching filaments with multiple rows of spherical to ovoid cocci, with a typical 'tram-track' appearance suggestive of Dermatophilus congolensis. Culture of the scab materials on sheep blood agar in the presence of 10% carbon dioxide yielded typical beta haemolytic colonies of D. congolensis from 75 samples. The isolates were further confirmed by the macroscopic and microscopic morphology of the colonies, and biochemical test results. This study confirmed the presence of dermatophilosis caused by D. congolensis in cattle in Kerala.

Simultaneous determination of cyclohexene oxide and its metabolites in rat plasma and urine by gas chromatography.

An assay was developed for the simultaneous measurement of cyclohexene oxide and its metabolites (cyclohexanol, trans-cyclohexane-1,2-diol, cyclohexane-1,2-diol-O-glucuronide, and N-acetyl-S-(2-hydroxycyclohexyl)-L-cysteine) in rat urine and plasma using gas chromatography. A mixture of ethyl acetate-acetonitrile (70:30) was used as the extracting solvent for both matrices. This liquid-liquid extraction procedure was followed by the separation of cyclohexene oxide and its metabolites on an HP-FFAP fused-silica capillary column. In order to determine the amount of cyclohexane-1,2-diol-O-glucuronide, samples were incubated at 37 degrees C with beta-glucuronidase and the amount of cyclohexane-1,2-diol formed from the reaction determined. The extraction efficiencies of cyclohexene oxide and cyclohexanol were greater than 90% both in urine and plasma. However, recovery from the plasma and urine for trans-cyclohexane-1,2-diol (60-68%) and N-acetyl-S-(2-hydroxycyclohexyl)-L-cysteine (approximately 76%) were considerably less. Long term stability studies showed that urine samples spiked with cyclohexene oxide and trans-cyclohexane-1,2-diol are stable at -20 degrees C for up to 9 weeks. However, plasma samples are only stable for up to 2 weeks under the same conditions. The calibration curves for all analytes were linear over the range of 12.5 to 400 micrograms/ml and correlation coefficients (r2) were greater than 0.990. The limit of detection for cyclohexene oxide, cyclohexanol, and N-acetyl-S-(2-hydroxycyclohexyl)-L-cysteine is 1.56 micrograms/ml, while the limit of detection for trans-cyclohexane-1,2-diol is 3.12 micrograms/ml. This method has been used for the determination of the disposition and metabolism of cyclohexene oxide, and may be applied in environmental monitoring, as well as in microbiological studies for other epoxide materials.

Absorption, disposition kinetics, and metabolic pathways of cyclohexene oxide in the male Fischer 344 rat and female B6C3F1 mouse.

Cyclohexene oxide (CHO) is a monomer intermediate used in the synthesis of pesticides, pharmaceuticals, and perfumes. Although CHO has a variety of industrial uses where direct human exposure is possible, very little is known about its fate in the body. Therefore, the objectives of this study were to determine the absorption, distribution, metabolism, and excretion of cyclohexene oxide after oral, intravenous, and dermal exposure in male Fischer 344 rats and female B6C3F, mice. After intravenous administration of [14C]CHO (50 mg/kg), CHO was rapidly distributed, metabolized, and excreted into the urine. Plasma concentrations of CHO rapidly declined and were below the limit of detection within 60 min. Average (+/- SD) values for terminal disposition half-life, apparent volume of distribution at steady-state, and systemic body clearance were: 19.3 +/- 1.6 min; 0.44 +/- 0.08 liter/kg; and 31.3 +/- 0.5 ml/kg * min, respectively. After oral administration of [14C]CHO (10 and 100 mg/kg), it was found that 14C-equivalents were rapidly excreted in the urine of both species. At 48 hr, the majority of the dose (73-93%) was recovered in urine, whereas fecal elimination accounted for only 2-5% of the dose. At no time after oral administration was parent CHO detected in the blood. However, its primary metabolite cyclohexane-1,2-diol was present for different lengths of time depending on the dose. Four metabolites were detected and identified in mouse urine by MS: cyclohexane-1,2-diol; cyclohexane-1,2-diol-O-glucuronide; N-acetyl-S-(2-hydroxycyclohexyl)-L-cysteine; and cyclohexane-1,2-diol-O-sulfate. The sulfate conjugate was not present in rat urine. Topical application of [14C]CHO (60 mg/kg) provided poor absorption in both species. The majority of 14C-equivalents applied dermally were recovered from the charcoal skin trap (approximately 90% of the dose). Only 4% of the dose was absorbed, and the major route of elimination was via the urine. To evaluate the toxicity of CHO, animals were given daily doses of CHO orally and topically for 28 days. No statistically significant changes in final body weights or relative organ weights were noted in rats or mice treated orally with CHO up to 100 mg/kg or up to 60 mg/kg when given topically. Very few lesions were found at necropsy, and none were considered compound related. In conclusion, regardless of route, CHO is rapidly eliminated and excreted into the urine. Furthermore, after either oral or dermal administration, it is unlikely that CHO reaches the systemic circulation intact due to its rapid metabolism, and is therefore unable to cause toxicity in the whole animal under the test conditions used in this study.

Absorption and disposition kinetics of 3,3',4,4'-tetrachloroazoxybenzene in the male Fischer 344 rat.

3,3',4,4'-Tetrachloroazoxybenzene (TCAOB) is a structural analog of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). It is formed as a byproduct during the synthesis of industrial products such as herbicides. TCAOB is a ligand for the Ah receptor and, at much higher doses, exhibits toxicities similar to TCDD. Although the reduced in vivo toxicity of TCAOB probably reflects differences in disposition, this study characterized its absorption and disposition kinetics. Male Fischer 344 rats were administered [14C]TCAOB (3.4 or 34 mg/kg p.o., 3.4 mg/kg i.v.), and the excretion of the radiolabel was monitored over 96 hr. After the low and high dose, 35% and 30% of the [14C]TCAOB were eliminated in the urine, with 55% and 54% eliminated in the feces. At 96 hr, the adipose tissue:blood ratios of [14C]TCAOB equivalents were 8 and 26 for the low and high doses, respectively. After the intravenous dose of TCAOB, the adipose tissue:blood ratio was 21 at 96 hr. Other tissue:blood ratios were of little significance (0.06-3.2). Pharmacokinetic parameters indicate that the parent molecule is cleared from blood with an average half-life of 7 hr and an average clearance of 11 ml/min.kg. Absolute bioavailability was calculated to be approximately 9%. Urine contained a variety of dichlorolaniline conjugates, which support the importance of azo reduction in the disposition of TCAOB. When compared with TCDD, the absorption of TCAOB is greatly reduced and the elimination of metabolites greatly enhanced. Therefore, at equal molar oral doses, TCAOB would express lower levels of Ah receptor-mediated toxicity than those defined for TCDD.

3,3',4,4'-tetrachloroazobenzene absorption, disposition, and metabolism in male Fischer 344 rats.

3,3',4,4'-Tetrachloroazobenzene (TCAB) is a contaminant generated during the synthesis of 3,4-dichloroaniline and 3,4-dichloroaniline-derived pesticides. TCAB is isosteric to 2,3,7,8-tetrachlorodibenzo-p-dioxin and has been shown to bind to the Ah receptor. Following oral administration of [14C]TCAB (3.2 and 32 mg/kg), 39-45% of the dosed radioactivity was excreted into the urine and 53-56% was recovered in the feces within 48 hr. Less than 6% of the dosed radioactivity remained in the tissues examined at 96 hr. After intravenous administration (3.2 mg/kg), 33% of the dose was excreted in the bile during 6 hr. TCAB metabolites in urine were identified using LC/MS. The major metabolites were sulfate ester conjugates of hydroxylated mono- or dichloroaniline derivatives. Some of these metabolites were also acetylated. After intravenous administration, the disappearance of [14C]TCAB from blood was monitored, and the pharmacokinetic profile was consistent with a two-compartment model. Pharmacokinetic parameters reveal that the compound is readily cleared from the blood with a t1/2 of 4.0 hr, clearance of 12.3 ml/min.kg, and an apparent volume of distribution of 4.3 liters/kg. The absolute oral bioavailability was determined to be 30%. The extensive azo reduction of TCAB decreases its systemic absorption after oral administration and thereby limits the amount of parent compound available to interact with the Ah receptor and decreases the Ah receptor-mediated toxicity.

Effect of bleomycin-induced fibrosis on pulmonary metabolism of selected xenobiotics.

Few studies are available characterizing the effects of fibrosis on pulmonary disposition of drugs or environmental pollutants. Two model substrates, p-nitroanisole and carbofuran, were selected to evaluate the effect of bleomycin-induced fibrosis on pulmonary disposition and metabolism using the isolated perfused lung and in vitro enzyme preparations. The rate of p-nitroanisole oxidation in the isolated perfused lung was significantly lower in fibrotic (k = 0.0334 min-1) than in control (k = 0.0493 min-1) lungs. However, there was no difference in the amount of p-nitrophenol formed between control (38 +/- 4 micrograms) and fibrotic (47 +/- 7 micrograms) lungs. Carbofuran clearance was similar in control (t1/2 = 91 min, ke = 0.008 min-1) and fibrotic (t1/2 = 75 min, ke = 0.009 min-1) lungs and was consistent with a one-compartment model. The Km value for p-nitrophenol formation in microsomes (0.185 +/- 0.095 mM) from control lungs was similar to fibrotic lungs (0.054 +/- 0.014 mM); however, Vmax was significantly higher in healthy (34.6 +/- 6.3 pmoles/min per mg microsomal protein) than in fibrotic (11.16 +/- 3.19 pmoles/min per mg microsomal protein) lungs. In vitro carbofuran studies indicated limited metabolism of carbofuran in both healthy and fibrotic microsomal enzyme preparations (< 5% of the administered dose). Lower p-nitroanisole metabolism in fibrotic lungs was consistent with lower levels of cytochrome P-450 2B1/B2 measured in bleomycin-treated lungs. Results suggest that individuals with bleomycin-induced pulmonary fibrosis may be at greater risk when exposed to certain toxic environmental chemicals or drugs that require detoxification by pulmonary microsomal enzymes.

Effect of tolbutamide and glyburide on pyruvate kinase flux in isolated rat hepatocytes.

The effects of two representative sulfonylureas, tolbutamide and glyburide, on pyruvate kinase (PK) flux were examined in fasted rat hepatocytes. PK flux was estimated by trapping 14C from NaH14CO3 in a 2 mM lactate pool, accounting for any incomplete trapping by parallel incubations with L-[1-14C]alanine. Glyburide (20 microM) and tolbutamide (1 mM) decreased glucose formation by 34.9% and 54.8%, respectively, from 2 mM lactate. This decrease in glucose formation was associated with a proportional decrease in pyruvate carboxylase (PCOX) flux (32.7% and 50.5%, respectively). Under these conditions, no net change in PK flux was observed. When hepatocytes were preincubated with lactate and/or sulfonylurea addition for 30 min prior to radiolabeling with NaH14CO3, the metabolic state of the cells changed markedly. Glyburide produced a 34.6% decrease in glucose formation and a 31.3% decrease in PCOX flux, but no change in PK flux. In contrast, tolbutamide decreased glucose formation by 12.5% and increased PK flux by 53.2%, but no change in PCOX flux was observed. Such an increase in PK flux may be linked to tolbutamide-mediated increases in fructose-1,6-bisphosphate (F16P) via fructose-2,6-bisphosphate (F26P). These findings demonstrate that tolbutamide and glyburide decrease hepatic glucose production through various alterations in carbohydrate metabolism, depending upon the metabolic state of the cell. In addition, F26P may play a larger role in the hypoglycemic mechanism of action of tolbutamide than glyburide, since pyruvate carboxylase accounted for most of the decrease in glucose formation observed with glyburide and because preincubation with tolbutamide resulted in an activation of PK.

The relationship of increased amniotic fluid volume to perinatal outcome.

We studied a group of 135 patients with polyhydramnios diagnosed on the basis of ultrasonic findings (greatest vertical diameter of the liquor column greater than or equal to 8.0 cm) between 32 and 36 weeks gestation (study group). We compared the obstetric characteristics and perinatal outcome of the study group with a similar number who constituted our control group. The incidence of women aged 20 years or less was higher in the study group (8.9%) compared with 4.5% in the control group. Of the 135 patients who were diagnosed to have polyhydramnios ultrasonically, the clinical diagnoses prior to referral for ultrasonic scanning, were, suspected large for date fetuses in 34 patients (25.2%), clinically suspected polyhydramnios in 28 (20.7%), gestational diabetes in 21 (15.6%) and insulin dependent diabetes in 6 (4.4%) compared with 13.3%, 5.2%, 3.0% and 0.7%, respectively in the control group (P less than 0.05). Preterm delivery occurred in 11.1% in the study group compared with the incidence of 6.7% in the control group. The incidence of fetal distress, low Apgar Score, macrosomic infants, major fetal anomalies, gross and corrected perinatal mortality rate and admission to special/intensive care nursery was significantly higher in the study group compared with that of the control (P less than 0.01). We found ultrasonic examination is a reliable technique to assess the amount of amniotic fluid volume and it alerts the clinician to possible future problems in pregnancy, labor and neonatal period.

Ultrasound evaluation of amniotic fluid: outcome of pregnancies with severe oligohydramnios.

We studied a group of 247 patients out of 7725 patients who had an ultrasonic examination between 32 and 36 weeks gestation and were found to have oligohydramnios (amniotic fluid volume (AFV) less than or equal to 2.0 cm). We compared the features of labor and mode of delivery and perinatal outcome in this group with that of a normal control group of 247 patients who also had an ultrasonic examination between 32 and 36 weeks gestation and were found to have normal AFV (greater than 2.0 to less than 8.0 cm). The incidence of induction of labor, of elective cesarean section, of preterm delivery (less than 37 weeks' gestation) was significantly higher in the oligohydramnios group (study group) as compared with the incidence in the control group (P less than 0.05). The incidence of fetal distress in antenatal and intrapartum period, pH (less than or equal to 7.2), of low Apgar score (0-5), of intrauterine growth retardation (IUGR) (less than tenth centile) infants, of major fetal anomaly and perinatal mortality rate (PNMR) are significantly higher in the oligohydramnios group as compared with the incidence and PNMR in the normal control group (P less than 0.05). The ultrasonic finding of oligohydramnios should alert the clinician regarding the possibility of problems in labor and perinatal period.

Ultrasonic assessment of ovulation in cyclofenil induced ovulatory cycles.

Graafian follicle growth was studied by ultrasound scanning during the peri-ovulatory period in 64 ovulatory cycles in 32 infertile patients on cyclofenil treatment, and compared with a control group of 32 patients with confirmed ovulatory cycles assessed on the basis of serum progesterone levels in the middle of the second half of the cycle. The mean maximum diameters of the leading follicles before ovulation were 21.9 +/- 0.6 (S.E.) mm and 24.4 +/- 0.5 (S.E.) mm, respectively for the cyclofenil group and the normal control group (P greater than 0.05). In 79% of the cyclofenil stimulated group and 83% of the spontaneous ovulation group, ultrasonic evidence of ovulation was present between 12 and 36 h after the initial increase in urine LH levels. Ultrasound scanning was found to be simple, and a quick method of monitoring graafian follicle development and ovulation on cyclofenil therapy and the cycles were comparable to the spontaneous ovulatory cycles as assessed on the basis of graafian follicle diameter, and the time of ovulation. Cervical score was not found to be useful to assess ovulation time in the cyclofenil treated group since 31.3% achieved a score of 10 or more on day -4, 93.8% within 24 h of ovulation and 24% on day 3 following the ovulation.

Ultrasonic assessment of cervix in "at risk" patients.

One hundred seventy patients were considered to be at risk of cervical incompetence from their previous history. They were scanned serially from the first trimester to 36 weeks gestation. Of the 170, 105 patients (61.8%) were found to have defective cervix (the length of cervix, width of the cervix, and cervical canal at the level of the internal cervical os, and herniation of amniotic membrane with or without fetal parts into the cervical canal). Of the 105 patients diagnosed to have defective cervix (possible cervical incompetence), 22 patients (21%) aborted, 48 patients (45.7%) had to have cervical cerclage, 26 patients (24.8%) had preterm delivery and the remaining 9 patients (8.5%) had no problems during pregnancy. The remaining 65 patients (38.2%) did not have ultrasonic evidence of cervical incompetence and 3 aborted and only 2 patients delivered between 35 and 37 weeks gestation while 60 patients delivered between 37 and 42 weeks gestation. These 65 patients would have had cervical cerclage on the basis of the clinical history alone, but in only 6, cervical cerclage was inserted.

Ultrasonic assessment of cervix in normal pregnancy.

We studied 30 normal pregnant women from the first trimester to 36 weeks. This group of women had had no previous miscarriage, termination of pregnancy, operations on the cervix, or premature labor. Fifteen were primigravidae and the remainder were multigravidae. There was no significant difference in the length of the cervix from 10 to 36 weeks of gestation (p greater than 0.05). The mean width of the cervix gradually increased from 10 to 36 weeks of gestation (p less than 0.01). The mean width of the cervical canal showed no significant change from 10 to 36 weeks of gestation (p greater than 0.05).