Khalid S. Ishaq

Alkyl-linked diglycerides inhibit protein kinase C activation by diacylglycerols

Alkylacylglycerols are synthesized when choline-phospholipids are degraded by a phospholipase C. This class of compounds has been shown to have biological activities; however, the mechanism of action is unknown. A series of alkyl-linked diglycerides were synthesized and tested for activity in an in vitro assay for protein kinase C. When protein kinase C activity was stimulated with the synthetic diacylglyceride analog 1-oleoyl-2-acetyl-sn-glycerol, the addition of alkyl glycerides caused a concentration-dependent inhibition of protein kinase C activity. Comparison of the protein kinase C inhibition by this series of 1-O-alkyl-2-acyl analogs revealed that both saturated and unsaturated long-chain groups in position 1 were effective and that dietherglycerols with short-chain moieties in position 2 were also effective. It is concluded from these studies that the biological activity of alkyl-linked glycerides may be expressed through protein kinase C inhibition.

Modification of glycerolipid metabolism in L-M fibroblasts by an unnatural amino-alcohol, N-isopropylethanolamine

Abstract The unnatural amino-alcohol, N-isopropylethanolamine, is incorporated into a phospholipid by monolayers of L-M fibroblasts. This phospholipid was identified as 1,2-diacyl- sn -glycero-3-phosphoisopropylethanolamine by using chemical and enzymatic procedures combined with thin-layer and gas-liquid chromatography. Since the phospho-N-isopropylethanolamine moiety is removed by phospholipase C, the stereochemistry of the phospholipid analog is identical to naturally occurring phosphoglycerides. Incubation of cells in 10 mM N-isopropylethanolamine inhibited the incorporation of [14C]choline and [14C]ethanolamine into phospholipids and stimulated the incorporation of [1-14C]palmitic acid and [1-14C]hexadecanol into triacylglycerols and alkyldiacylglycerols. These results indicate that N-isopropylethanolamine affects glycerolipid synthesis at the diradylglycerol branch point.

Incorporation and subcellular distribution of an unnatural phospholipid base-analog, N-isopropylethanolamine, in rat liver

An unnatural phospholipid, phosphatidyl-N-isopropylethanolamine, was isolated from rat liver after intraperitoneal injections of N-isopropylethanol-amine; it was identified on the basis of enzymic, chemical, and chromatographic analyses. Although this phospholipid was formed at the expense of phosphatidylcholine and phosphatidylethanolamine, its fatty acid composition did not resemble either of these lipids. Microsomes, mitochondria, and plasma membranes contained significant amounts (up to 9%) of this unusual phospholipid. Radioisotope incorporation experiments suggest that the N-isopropylethanol-amine containing phospholipid is rapidly equilibrated between microsomes and mitochondria and more slowly with surface membranes.

Phospholipid analogs against HIV-1 infection and disease

Phospholipid analogs are a new class of compounds with potent activity against HIV infection when used alone or conjugated with other therapeutic agents. When conjugated to the nucleoside analog AZT, the resulting phospholipid-AZT conjugate can double target the virus replication cycle by inhibiting the viral reverse transcriptase (by AZT) and inducing the production of defective virus particles that lack functional gp120 expression on the virus surface resulting in reduced capacity to bind to CD4+ cells and inhibition of infected cell-cell fusion (by phospholipid). Of great interest are data indicating that selected phospholipids are active against drug resistant variants, a current major problem in treating HIV/AIDS and controlling the epidemic occurring in various parts of the world. The purpose of this review is to provide current information on the design and synthesis of various types of phospholipids and phospholipid conjugates, in-vitro and in-vivo antiviral activity, tissue distribution, intracellular metabolism, and mechanism of action. The future development of this novel class of compounds offers an exciting approach for reducing the toxicity and enhancing the distribution of therapeutic drugs to the lymphatics and central nervous system and suppressing the emergence of drug resistant variants of HIV.

Boronic Acid Derivatives Targeting HIV-1

A series of novel boronic acid derivatives containing either a pyrimidine or purine base was synthesized. The preparation involved the condensation of 4-bromobutyl boronic acid with the appropriate base. These acyclic nucleosides were designed as potential antiviral agents especially targeting the human immunodeficiency virus. Two analogues, 6-chloro-9-(4-dihydroxyborylbutyl)purine and 2,6-dichloro-9-(4-dihydroxyborylbutyl)purine, exhibited EC50 values of 7.7 μM and 0.99 μM, respectively, in an HIV-1 syncytial plaque reduction assay.

Compounds that inhibit chymotrypsin and cell replication

Several compounds have been tested for their ability to inhibit bovine pancreatic alpha-chymotrypsin (Ki) and their ability to inhibit cell replication (IC50). There is good agreement over three orders of magnitude between the Ki and the IC50 values of these compounds. The data support the hypothesis that a cellular, chymotrypsin-like activity is necessary for cell replication.

Molecular simulation of alkyl boronic acids: molecular mechanics and solvation free energy calculations

The alkyl boronic acid moiety is incorporated into many biologically interesting structures. To provide parameters for molecular mechanics and dynamics studies of compounds containing this group, we performed ab initio calculations at the 6‐31G* level to obtain bond stretching, bending, and torsion constants. The hydrodynamic formulation of the time‐dependent density functional theory was used to calculate the attractive part of van der Waals (VDW) 6–12 potential. The geometry of boronic acid moiety of the 6‐31G* optimized methyl boronic acid was similar to that of the X‐ray crystal structure of phenyl boronic acid. To test the reliability of nonbonded parameters, Monte Carlo free energy perturbation simulations and the thermodynamic cycle approach were used to estimate the differences in solvation free energy between alkyl alcohol and alkyl boronic acid, both in water and in chloroform. These free energy differences were also obtained experimentally by measuring the vapor–water and water–chloroform partition coefficients. The close agreement between experimental values and the results of our simulations suggests the reliability of new molecular mechanics force‐field parameters for alkyl boronic acids.

The inhibition of rat liver chromatin protease by congeners of the phenylboronic acids.

A group of arylalkylboronic acids were synthesized in order to investigate the inhibitory potential of these compounds for rat liver chromatin protease (EC 3.4--). The effect of side chain length, side chain substitution and aromatic substitution on proteolytic activity in chromatin dissociated in salt and urea was assayed. It was determined that a side chain length two carbons long provided the greatest inhibitory effect with complete inhibition attainable at 20 mM concentration of phenylethylboronic acid. Aryl substitution in the ortho position proved to be the most potent structural change with complete inhibition attained by 1 mM concentration of 0-methylphenylethylboronic acid. The binding of these two inhibitors proved to be reversible.

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 Biological Evaluation of Ether-Linked Derivatives of Phosphatidylinositol

The synthesis of two novel glycero-3-phosphoinositol ether lipid analogues, rac-l-O-octadecyl-2-O methylglycero-3-phospho-myo-inositol 6 (an ether lipid analogue of rac-l-O-octadecyl-2-O-methylglycero-3-phosphocholine; ET-18-OMe) and rac-l-O-octadecyl-2-O-acetylglycero-3-phospho-myo-inositol 11 (an ether lipid analogue of platelet-activating factor), is described. The two target compounds and the synthetic intermediates were evaluated for inhibition of HL60, BG1, and BG3 human malignant cells in vitro and inhibition of protein kinase C. Tumor inhibitory activity was found for compounds 6 and 11 in all systems but not for their synthetic intermediates. However, compounds 6 and 11 as well as the synthetic intermediates 5 and 13, but not 9, exhibited protein kinase C inhibitory activity.