In-silico and in-vitro screening of Asiatic acid and Asiaticoside A against Cathepsin S enzyme.

BACKGROUND: Atherosclerosis is a form of cardiovascular disease that affects the endothelium of the blood vessel. Series of events are involved in the pathophysiology of this disease which includes the breaking down of the connective tissue elastin and collagen responsible for the tensile strength of the arterial wall by proteolytic enzyme. One of these enzymes called Cathepsin S (CatS) is upregulated in the progression of the disease and its inhibition has been proposed to be a promising pharmacological target to improve the prognosis of the disease condition. Asiatic acid and asiaticoside A are both pentacyclic triterpenoids isolated from Centella asiatica. Their use in treating various cardiovascular diseases has been reported. METHODS: In this study through in silico and in vitro methods, the pharmacokinetic properties, residue interaction, and inhibitory activities of these compounds were checked against the CatS enzyme. The SwissADME online package and the ToxTree 3.01 version of the offline software were used to determine the physicochemical properties of the compounds. RESULT: Asiatic acid reported no violation of the Lipinski rule while asiaticoside A violated the rule with regards to its molecular structure and size. The molecular docking was done using Molecular Operating Environment (MOE) and the S-score of - 7.25988, - 7.08466, and - 4.147913 Kcal/mol were recorded for LY300328, asiaticoside A, and asiatic acid respectively. Asiaticoside A has a docking score value (- 7.08466Kcal/mol) close to the co-crystallize compound. Apart from the close docking score, the amino acid residue glycine69 and asparagine163 both interact with the co-crystallized compound and asiaticoside A. The in vitro result clearly shows the inhibitory effect of asiaticoside and asiatic acid. Asiaticoside A has an inhibitory value of about 40% and asiatic acid has an inhibitory value of about 20%. CONCLUSION: This clearly shows that asiaticoside will be a better drug candidate than asiatic acid in inhibiting the CatS enzyme for the purpose of improving the outcome of atherosclerosis. However, certain modifications need to be made to the structural make-up of asiaticoside A to improve its pharmacokinetics properties.

Proprotein convertase subtilisin/kexin type 9 genetic screening using the vervet (Chlorocebus aethiops) model.

BACKGROUND: The proprotein convertase subtilisin/kexin type 9 (PCSK9) gene has come to prominence due to its reported function in the clearance of low-density lipoprotein cholesterol. The vervet monkey (Chlorocebus aethiops) was utilized to study the genetics of PCSK9 gene. METHOD: Sixteen vervet monkeys were selected to screen for possible PCSK9 polymorphisms and to determine gene expression. RESULTS: Four PCSK9 sequence variants (T112T, R148S, H177N and G635G) were identified and three of these variants (H177N, R148S, and G635G) were categorized as loss of function mutations. A decline in gene expression levels was also observed in animals harboring these three variants. Although the selected variants might have affected the level of gene expression in the selected animals, individual variation was also noticed in some of these individuals with the G635G variant. CONCLUSION: Based on the findings obtained from this study, it is suggestive that the activity of PCSK9 was hindered.

The expression of type I and II gonadotropin-releasing hormone receptors transcripts in Vervet monkey (Chlorocebus aethiops) spermatozoa.

Gonadotropin-Releasing Hormone (GnRH), acting via the GnRH receptor (GnRHR), and a member of G-protein coupled receptor (GPCR), plays an essential role in the control of reproduction while operating primarily at the hypothalamic level of the gonadotropic axis. GnRH and its receptor are co-expressed in certain specific cells, suggesting an autocrine regulation of such cells. In the male reproductive system, two forms of GnRH (I and II) and its receptors (GnRHR) are present in the human and non-human primate (NHP) testis, prostate, epididymis, seminal vesicle, and human spermatozoa. In humans, the GnRHR-II receptor gene is disrupted by a frameshift in exon 1 and a stop codon in exon 2, rendering the receptor non-functional, whereas a fully functional GnRHR-II receptor is present in New-World and Old-World monkeys. There is no evidence of the existence of a GnRH receptor in NHP sperm. Since the NHP has a phylogenetic relationship to man and is often used as models in reproductive physiology, this present study aimed to determine GnRHR-I and GnRHR-II in Vervet monkey (Chlorocebus aethiops) spermatozoa. A total of 24 semen samples were obtained from four adult Vervet monkeys through electro-ejaculation and utilized for genotyping and gene expression analysis of GnRHR-I and II. Here we report that both receptors were successfully identified in the Vervet monkey sperm with the abundance of GnRHR-I gene expression compared to GnRHR-II. In comparison to the human, there is no evidence of such a stop codon at position 179 in exon 2 of the Vervet GnRHR-II. These findings suggest that both receptors are transcriptionally functional in Vervet spermatozoa.

Mutational analysis of BFSP1, CRYBB1, GALK1, and GJA8 in captive-bred vervet monkeys (Chlorocebus aethiops).

BACKGROUND: Congenital cataract has been reported in a colony of captive-bred vervet monkeys (Chlorocebus aethiops). METHODS: Molecular tools such as genotyping and gene expression were used to identify mutations associated with congenital cataract in this vervet colony. Beaded filament structural protein 1 (BFSP1), beta-crystallin B1 (CRYBB1), galactokinase1 (GALK1), and gap junction alpha-8 protein (GJA8) were screened, sequenced, and analyzed for mutations in 24 vervet monkeys (control and cataract). RESULTS: Five missense sequence variants were identified (V147E, A167P, L212F, N55K, and T247A), three of which were found to be potentially disease-causing. Furthermore, downregulation was observed in BFSP1, CRYBB1, and GALK1 genes. CONCLUSION: This study reports two cases of incomplete penetrance and/or uniparental disomy (L212F and T247A) in BSFP1. Mutations in BSFP1 together with three mutations in GALK1 and GJA8 were predicted to be disease-causing.

The effect of hyperglycinemic treatment in captive-bred Vervet monkeys (Chlorocebus aethiops).

Nonketotic hyperglycinemia (NKH) is a neuro-metabolic disorder caused by a deficiency in the glycine cleavage system (GCS) and glycine transporter 1 (GlyT1). A case of atypical late onset of NKH has been reported in a colony of captive-bred Vervet monkeys. The purpose of this study was to evaluate the effect of sodium benzoate and dextromethorphan in reducing glycine levels in hyperglycinemic monkeys. Twelve captive-bred Vervet monkeys were assigned into three groups consisting of four animals (control, valproate induced and cataract with spontaneous hyperglycinemia). Valproate was used to elevate glycine levels and the induced group was then treated with sodium benzoate and dextromethorphan together with group three to normalise glycine levels in cerebrospinal fluid (CSF) and plasma. Valproate induction elicited changes in phosphate, alkaline phosphatase and platelet count, however, no significant changes in the glycine levels were observed, and this might be due to the individual variability within the group. The treatment intervention was only obtained in the spontaneous group whereby the glycine levels were normalised in CSF and plasma. Therefore, it can be concluded that sodium benzoate and dextromethorphan treatment was effective and beneficial to the hyperglycinemic group.

Nonketotic hyperglycinemia in captive-bred Vervet monkeys (Chlorocebus aethiops) with cataracts.

BACKGROUND: Nonketotic hyperglycinemia (NKH) is a rare metabolic disorder that is characterized by high levels of glycine in plasma and cerebrospinal fluid in humans. In this study, total congenital cataract captive-bred Vervet monkeys (Chlorocebus aethiops) that are hyperglycinemic were screened to identify mutations in Bola type 3 (BOLA3), glutaredoxin 5 (GLRX5), and lipoate synthase (LIAS) genes. METHODS: Twenty-four Vervet monkeys (12 hyperglycinemic and 12 healthy controls) were selected for mutation analysis using polymerase chain reaction (PCR), Sanger sequencing, and reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: Novel sequence variants were identified in BOLA3 (R23H and Q38R) and LIAS (R369I and A371A), and gene expression in the control group was significantly lower compared to the hyperglycinemic group (P < 0.05). CONCLUSION: The data obtained from this study will contribute to generation of new knowledge regarding the involvement of these genes in NKH development.

Autosomal recessive congenital cataract in captive-bred vervet monkeys (Chlorocebus aethiops).

BACKGROUND: The aim of the study was to evaluate the genetic predisposition of congenital cataract in a colony of captive-bred vervet monkeys. METHODS: Four congenital cataract genes: glucosaminyl (N-acetyl) transferase 2 (GCNT2), heat shock transcription factor 4 (HSF4), crystallin alpha A (CRYAA) and lens intrinsic membrane protein-2 (LIM2) were screened, sequenced and analysed for possible genetic variants in 36 monkeys. Gene expression was also evaluated in these genes. RESULTS: Fifteen sequence variants were identified in the coding regions of three genes (GCNT2, HSF4 and CRYAA). Of these variations, only three were missense mutations (M258V, V16I and S24N) and identified in the GCNT2 transcripts A, B and C, respectively, which resulted in a downregulated gene expression. CONCLUSION: Although the three missense mutations in GCNT2 have a benign effect, a possibility exists that the candidate genes (GCNT2, HSF4 and CRYAA) might harbour mutations that are responsible for total congenital cataract.

Mutation analysis of GLDC, AMT and GCSH in cataract captive-bred vervet monkeys (Chlorocebus aethiops).

BACKGROUND: Non-ketotic hyperglycinaemia (NKH) is an autosomal recessive inborn error of glycine metabolism characterized by accumulation of glycine in body fluids and various neurological symptoms. METHODS: This study describes the first screening of NKH in cataract captive-bred vervet monkeys (Chlorocebus aethiops). Glycine dehydrogenase (GLDC), aminomethyltransferase (AMT) and glycine cleavage system H protein (GCSH) were prioritized. RESULTS: Mutation analysis of the complete coding sequence of GLDC and AMT revealed six novel single-base substitutions, of which three were non-synonymous missense and three were silent nucleotide changes. CONCLUSION: Although deleterious effects of the three amino acid substitutions were not evaluated, one substitution of GLDC gene (S44R) could be disease-causing because of its drastic amino acid change, affecting amino acids conserved in different primate species. This study confirms the diagnosis of NKH for the first time in vervet monkeys with cataracts.