Nathan Iyer

In vitro evaluation and characterization of newly designed alkylamidophospholipid analogues as anti-human immunodeficiency virus type 1 agents

Our laboratories first reported two novel classes of complex synthetic lipids, including alkylamidophosphocholines (PC lipid; CP-51) and alkylamidophosphate ester-linked lipid–AZT conjugates (lipid–AZT conjugates; CP-92), with selective and potent activity against human immunodeficiency virus type 1 (HIV-1). To extend these observations, we synthesized additional PC lipids and lipid–AZT conjugates (INK and INK–AZT conjugate) to evaluate their structure–activity relationships by testing for selectivity against infectious wild-type (wt) and drug-resistant HIV-1 replication, virus fusogenic activity and toxicity for mouse bone marrow cells. PC lipid compounds with medium chain lengths at positions 1 and 2 gave an improved selective index (SI). INK-3, with 12 and 8 carbons and INK-15, with 10 and 12 carbons were among the most selective when evaluated in CEM-SS cells. INK-14, a lipid–AZT conjugate where AZT replaced the choline in PC lipid INK-3, gave the highest SI of >1250 against both infectious wt HIV-1 replication in CEM-SS cells and a clinical isolate in peripheral blood leukocytes. Notably, the PC lipid compounds INK-3 and INK-15, but not the lipid–AZT conjugate INK-14, were potent inhibitors of matched pairs of AZT-sensitive and AZT-resistant HIV-1 clinical isolates. INK-3 also inhibited replication of HIV-2 and TIBO-resistant HIV-1, and inhibited HIV-1-mediated fusogenic activity by 78, 41 and 9% in a dose-dependent manner. The TC50 for mouse bone marrow cells was >100 μg/ml for INK-3 compared to 9.15–14.17 μg/ml for CP-51 and 0.142–0.259 μg/ml for AZT. These data suggest that optimum PC lipid compounds are significantly less toxic than AZT and have high potential as novel therapeutic agents for AIDS.

Synthesis and Evaluation of a Novel Synthetic Phosphocholine Lipid‐AZT Conjugate That Double‐Targets Wild‐Type and Drug Resistant Variants of HIV

INK‐20, a synthetic phosphocholine lipid‐AZT conjugate, was evaluated for antiviral activity against wild‐type HIV‐1, a matched pair of pre‐AZT and post‐AZT and multi‐drug resistant clinical isolates. In addition, it was tested for activity against viruses resistant to nucleoside (AZT, 3TC) and nonnucleoside (nevirapine) reverse transcriptase and protease (saquinavir) inhibitors using the syncytial plaque reduction assay for infectious virus multiplication. The EC50 values were 0.004, and 0.005 µM against wild‐type HIV‐1 for INK‐20 and AZT, respectively. INK‐20 showed little or no cytotoxicity when assayed in CEM‐SS cells and four other cell types including PBMC. This resulted in a selective index of > 25,000 and > 20,000 for INK‐20 and AZT, respectively. When tested against a matched pair of pre‐AZT and post‐AZT clinical isolates, the EC50 values were 0.01 and 0.03 µM for INK‐20 and 0.0005 and 0.33 µM for AZT, respectively. INK‐20 had moderate to good activity against two other AZT resistant variants and very good activity against a multi‐drug resistant clinical isolate compared to marked resistance of these viruses to AZT alone. INK‐20 retained significant activity against viruses resistant to 3TC, nevirapine, and saquinavir. The synthetic phosphocholine lipid‐AZT conjugate INK‐20 represents a novel class of anti‐HIV compounds, which may provide new strategies for the treatment of HIV drug‐resistant variants. †In honor and celebration of the 70th birthday of Professor Leroy B. Townsend.

Amplified Assay for Specific Dual-Labeled DNA Using the Coagulation Cascade (EDNA-ELCA)

Detection of the products of the PCR reaction using nonisotopically labeled DNA molecules containing biotin, fluorescein, or digoxigenin has become a popular method for identification of specific products of polymerase chain reaction (PCR) (1,3). These labeled molecules are prepared either by PCR synthesis in the presence of labeled deoxyuridine triphosphate (1,3) or by hybridization of labeled probes to the unlabeled PCR product (1,2). Detection is then in a format very similar to enzyme-linked immunosorbent assays (ELISA) using similarly labeled antigens and antibodies, i.e., by capture on the receptor for one ligand (streptavidin or antibody) and using the other ligand for detection.