Reshu Saxena

One pot efficient diversity oriented synthesis of polyfunctional styryl thiazolopyrimidines and their bio-evaluation as antimalarial and anti-HIV agents.

An efficient one pot synthesis of a series of pluripotent (E)-1-(3-methyl-5-aryl-7-styryl-5H-thiazolo[3,2-a]pyrimidin-6-yl)-3-arylprop-2-en-1-ones is reported. It involves reaction of 5-acetyl-6-methyl-4-aryl-dihydropyrimidine-2-thiones, propargyl bromide and aromatic aldehydes in presence of ethanolic KOH. The newly synthesized compounds were evaluated for antimalarial activity against Plasmodium falciparum and as HIV-RT inhibitors. Most of the compound displayed potent antimalarial activity with IC(50)<2 μg/mL. Compounds 6, 11 and 20 showed better activity against P. falciparum K1 strains in comparison to standard drug chloroquine. Compounds 6, 11, and 16 exhibited 73.44, 66.92, and 70.81% HIV-RT inhibition at 100 μg/mL.

Human Beta Casein Fragment (54–59) Modulates M. bovis BCG Survival and Basic Transcription Factor 3 (BTF3) Expression in THP-1 Cell Line

Immunostimulatory peptides potentiate the immune system of the host and are being used as a viable adjunct to established therapeutic modalities in treatment of cancer and microbial infections. Several peptides derived from milk protein have been reported to induce immunostimulatory activity. Human β -casein fragment (54–59), natural sequence peptide (NS) carrying the Val-Glu-Pro-Ile-Pro-Tyr amino acid residues, was reported to activate the macrophages and impart potent immunostimulatory activity. In present study, we found that this peptide increases the clearance of M. bovis BCG from THP-1 cell line in vitro. The key biomolecules, involved in the clearance of BCG from macrophage like, nitric oxide, pro-inflammatory cytokines and chemokines, were not found to be significantly altered after peptide treatment in comparison to the untreated control. Using proteomic approach we found that BTF3a, an isoform of the Basic Transcription Factor, BTF3, was down regulated in THP-1 cell line after peptide treatment. This was reconfirmed by real time RT-PCR and western blotting. We report the BTF3a as a novel target of this hexapeptide. Based on the earlier findings and the results from the present studies, we suggest that the down regulation of BTF3a following the peptide treatment may augment the M. bovis BCG mediated apoptosis resulting in enhanced clearance of M. bovis BCG from THP-1 cell line.

Proteomic Profiling of SupT1 Cells Reveal Modulation of Host Proteins by HIV-1 Nef Variants

Nef is an accessory viral protein that promotes HIV-1 replication, facilitating alterations in cellular pathways via multiple protein-protein interactions. The advent of proteomics has expanded the focus on better identification of novel molecular pathways regulating disease progression. In this study, nef was sequenced from randomly selected patients, however, sequence variability identified did not elicited any specific mutation that could have segregated HIV-1 patients in different stages of disease progression. To explore the difference in Nef functionality based on sequence variability we used proteomics approach. Proteomic profiling was done to compare the effect of Nef variants in host cell protein expression. 2DGE in control and Nef transfected SupT1 cells demonstrated several differentially expressed proteins. Fourteen protein spots were detected with more than 1.5 fold difference. Significant down regulation was seen in six unique protein spots in the Nef treated cells. Proteins were identified as Cyclophilin A, EIF5A-1 isoform B, Rho GDI 1 isoform a, VDAC1, OTUB1 and α-enolase isoform 1 (ENO1) through LC-MS/MS. The differential expression of the 6 proteins was analyzed by Real time PCR, Western blotting and Immunofluorescence studies with two Nef variants (RP14 and RP01) in SupT1 cells. There was contrasting difference between the effect of these Nef variants upon the expression of these six proteins. Downregulation of α-enolase (ENO1), VDAC1 and OTUB1 was more significant by Nef RP01 whereas Cyclophilin A and RhoGDI were found to be more downregulated by Nef RP14. This difference in Nef variants upon host protein expression was also studied through a site directed mutant of Nef RP01 (55AAAAAAA61) and the effect was found to be reversed. Deciphering the role of these proteins mediated by Nef variants will open a new avenue of research in understanding Nef mediated pathogenesis. Overall study determines modulation of cellular protein expression in T cells by HIV-1 Nef variants.

Toxicity of a Novel Herbomineral Preparation Las01 on Human Cancer Cell Lines and Its Safety Profile in Humans and Animals

Polyhedral formulations based on Rasayana therapy described in Charaka Samhita showed remarkable improvement in quality of life of various cancer patients who have been found to be refractory or poor responders to modern chemotherapy and radiation treatment. One of the most recent novel herbomineral preparation, Las01 prepared absolutely as per the instruction given in the ancient Ayurvedic literature has been found to be effective as a potent anticancer drug in the human cell lines, the MCF-7 and Hela cancer cell lines. This novel preparation of Las01 is also found to be devoid of toxicity both in animals as well as in human subjects, which is the main drawback of chemotherapeutic agents used in modern system of medicine. Our results warrant multicentric clinical trials on a large scale which seems to be a future promising drug to cure incurables cancer patients.

PA01.43. In-vitro cyto chemical & flow-cytometry studies with las02- a coded herbo-mineral compound

Purpose: The drug optimization and understanding the mechanisms of action of drugs on the deregulation of cell cycle which is frequently considered as the cause of progression in cancer can provide important insights for new cancer treatment strategies. The drug LAS02 is a herbo mineral drug prepared as per ancient Ayurvedic literature. Method: In this study the effect of LAS02 was studied by analyzing the effect on cell cycle by flow cytometery on cancer cell lines breast cancer (MCF7), cervical cancer (HELA), colon cancer (COLO 205) and prostate cancer (DU 145), procured from NCCS, Pune. The cells were treated by different doses of LAS02, and assay for proliferation was performed by MTT assay test, subsequently, these were analyzed by flow cytometer for cell cycle analysis. Result: The results showed inhibition of proliferation in MCF7 by 77% and HELA cells by 78% at dose of 500μg/ml in MTT assay. In cell cycle analysis for COLO 205 treated with LAS02, the percentage retention of the cells in G0/ G1 phase was 73.07% at 300μg/ml as compared to 52.16% in the control after 24hrs. In DU 145, treated with LAS02, cells that retained at G0/G1 phase were 79.28% at a dose of 400μg/ml after 48hrs; as compared to control of 62.41%. The apoptosis observed at 400μg/ml drug concentration was 43.51%. Conclusion: The study shows that LAS02 acts as a potent anti cancerous compound by inhibiting proliferation as well as by inducing retention of cells in G0/G1 phase along with apoptosis significantly at in vitro level. Therefore, LAS02 arrests the cancerous cells in G0/G1 phase and prevented the entry of pre cancerous stem cells from G0/G1 phase into G2, the subsequent proliferative stage and inhibits cancer cells from completing the cell cycle. Such a finding is unique with this new drug, which holds a great promise as one of the most effective and safest cancerostatic drug.

OA01.46. Effect of LAS-02, a cancero-static compound on p53 levels in cases of different types of cancers

Purpose: Cancer is one of the life threatening diseases spreading worldwide now a days. Oncogenes on one hand are well known to be activated by proto-oncogenes and are suppressed by tumor suppressor genes (TSG). There are different strategies for targeting to the regulation of tumor suppression gene. P53 is a most targeted gene that regulates the cell cycle and hence functions as tumor suppression to maintain the integrity of DNA. Method: In the present study p53 was used as a parameter to evaluate the efficacy of a new herbo-mineral compound LAS02 a cancero-static compound formulated by Lavanya Ayurveda Hospital and Research Centre Lucknow, in different type of malignancies such as breast cancer, bone marrow, hepatic, ovarian, head & neck carcinoma. The serum samples were taken from the cancer patients at Lavanya Ayurvedic hospital and was tested by solid phase sandwich enzyme linked immuno-sorbent assay (ELISA). Result: The main p53 valves before treatment more found to be 4.76 U/ml however, after treatment with our new herbo-mineral drug, the mean values of post treated p53 values were found to be significantly raised to 35.94 U/ml, which signifies the efficacy of treatment of LAS02 in enhancing the expression of p53 gene in different types of cancers. Conclusion: p53 protein is a guard of DNA and helps in enhancing repair of the damage of DNA done by cancerous pathology.

PA01.08. An evaluation of a new anti-aids herbo-mineral compound (laa-1).

Purpose: AIDS is the deadliest disease of the decade, for which unfortunately even after several advancement in biomedical sciences, no curative treatment is available as no drug is capable of separating and recovering the motif between retro viral DNA of the host. Method: The present study has been undertaken to evaluate the role of a new herbo mineral compound (LAA01) in 18 cases suffering from AIDS after getting clearance from the Institutional Ethical committee (IEC). Criteria for assessing antiviral activity includes CD3, CD4, CD8, CD4/ CD8 ratio and viral load parameters were measured. These parameters were conducted at Biognosis an approved GLP lab in Hyderabad. Result: Pre treatment mean values of these parameters compared with LAA01 treated cases values periodically respectively given in following table. The results indicate a significant rise in CD4 counts and a significant decrease in viral load. Conclusion: It concludes that the drug Ayurvedic herbo mineral preparation LAA01 under test is not only a viridicidal (drug inhibiting the HIV replication) as is evident from decrease in HIV load, but the drug also enhanced immunity as is evident from increases in CD4 and CD8 counts and their respective percentage, which maintained the quality of life (QOL) of the patients, which needs further extensive clinical trial.