D. Kinchington

The Activity of Candidate Virucidal Agents, Low pH and Genital Secretions against HIV-1 In Vitro

Summary: The effect of low pH, normally present in the female genital tract, on HIV viability was examined. HIV is more acid stable than previously reported with no substantial reduction in infectivity occurring until pH levels are reduced below 4.5. The virucidal activity of 3 topical spermicides and chlorhexidine was assessed in vitro using previously established and newly modified assay systems. None of the agents tested had a selectivity index (SI) greater than 5.2. Semen and cervical secretions were assessed for their ability to inhibit HIV-1. While no virucidal effect was found in the latter, seminal fluid was found to have significant activity against HIV-1 and a SI of approximately 50.

A comparison of gag, pol and rev antisense oligodeoxynucleotides as inhibitors of HIV-1

Sequences from the gag, pol and rev regions of the RF strain of HIV-1 (HIV-1RF) were chosen as targets for antisense phosphorothioate oligodeoxynucleotides (S-oligos). These sequences were the p18/p24 junction in gag, the active site of HIV protease in pol; a sequence from the first exon of the rev gene and S-oligodeoxycytidylic acid controls. Compounds were tested against HIV-1 in both acutely and chronically infected cells. The results show that these phosphorothioate analogues tested in acutely infected cells were active in the 0.1-2 microM range, were dependent on chain length but had no sequence specificity. To study the mechanism of action, the time of addition of S-oligos to acutely infected cells was delayed for up to 48 h post-infection. It was found that antiviral activity was lost when compounds were added to the cultures later than 10 h post-infection. With chronically infected cells only the antisense rev sequence showed activity at 30 microM and neither of the gag or pol antisense sequences has a significant effect on HIV replication at 50 microM. These results are consistent with previous in vitro studies which demonstrate that antisense S-oligodeoxynucleotides have several modes of action.

Synthesis and anti-HIV Activity of Some Novel Substituted Dialkyl Phosphate Derivatives of AZT and ddCyd

The reaction of thymidine, AZT (Fig. 1, compound 1) and ddCyd (Fig. 1, compound 2) with bis(2,2,2-trichloroethyl) phosphorochloridate gave novel 5′-bis(2,2,2-trichloroethyl) phosphates, characterized by spectroscopic and analytical data. Analogous reactions with bis(2,2,2-trifluoroethyl) phosphorochloridate gave the corresponding AZT and ddCyd derivatives. In both of the ddCyd reactions, by-products were isolated and characterized as O5′, W4-diphosphorylated materials. It was hoped that the 5′-phosphate triesters might act as membrane-soluble pro-drugs of the bio-active free nucleotides of AZT or ddCyd. In fact, all of the 5′-phosphate derivatives of AZT and ddCyd displayed anti-HIV activity in vitro. Surprisingly, the thymidine compound also displayed very slight anti-HIV activity. The striking activity of the AZT and ddCyd derivatives is attributed to the metabolic instability of the substituted trialkyl phosphate moiety.

Phosphoramidate Derivatives of AZT as Inhibitors of HIV: Studies on the Carboxyl Terminus

Novel phosphoramidate derivatives of the anti-HIV nucleoside analogue AZT have been prepared by phosphorochloridate chemistry. These materials carry carboxy-protected, amino acids, and are designed to act as membrane-soluble prodrugs of the bio-active free nucleotides. In vitro evaluation revealed the compounds to have a pronounced, selective antiviral activity. In particular, variation in the carboxy terminus region is studied. For alkyl phosphates small changes in the structure of the amino ester lead to marked changes in biological activity. However, for analogous aryl phosphates there is little dependence on the structure of the ester. This suggests a different mechanism of action for these two categories of phosphate prodrug.

Nucleoside analogues previously found to be inactive against HIV may be activated by simple chemical phosphorylation

Nucleoside analogues previously found to be inactive against the human immunodeficiency virus (HIV) may be activated by simple chemical derivatisation. As part of our effort to deliver masked phosphates inside living cells we have discovered that certain phosphate triester derivatives of inactive nucleoside analogues become inhibitors of HIV replication. This discovery underlies the importance of the masked phosphate approach, and has significant implications for the future design of chemotherapeutic nucleoside analogues. If highly modified nucleoside analogues may be active without the intervention of nucleoside kinase enzymes, major advantage may accrue in terms of low toxicity and enhanced selectivity. Moreover, the increased structural freedom may have implications for dealing with the emergence of resistance. The concept herein described as ‘kinase bypass’ may thus stimulate the discovery of a new generation of antiviral agents.

Comparison of antiviral effects of zidovudine phosphoramidate and phosphorodiamidate derivatives against HIV and ULV in vitro

A number of zidovudine phosphoramidate and phosphorodiamidate derivatives were prepared, including some previously unreported benzyl esterified amino acyl compounds. These were found to be active in vitro against the human immunodeficiency virus (HIV-1), and were tested subsequently in the S+L-tissue culture assay against urethane leukaemia virus (ULV), a murine leukaemia virus (MuLV). The fifteen compounds tested showed a similar range of activity against the two viruses. No active compounds were missed in the MuLV system which was usually more sensitive to antiviral effects. Five compounds showed some toxicity to the mouse cells only. We are using this system in parallel with HIV assays to identify those derivatives which will be tested subsequently against a murine retrovirus in vivo.

Cytotoxicity of 3,4‐dihalogenated 2(5H)‐furanones

Mucohalogen acids have been used for the preparation of a variety of 3,4‐dihalogenated 2(5H)‐furanones. In one synthetic step the carbamates 2a‐c and the pseudoanhydrides 4a‐e were prepared using isocyanates and acid anhydrides. A series of 5‐alkoxylated 3,4‐dichloro‐2(5H)‐furanones 5a‐o have been synthesized with a wide range of lipophilicity, using the hydroxy‐form of mucohalogen acids 1a and 1b. The 5‐allyl‐3,4‐dichloro‐2(5H)‐furanone 5f was derived into the dihydro‐isoxazol 6 and the oxirane 7. The methyl ester 5a was converted with ammonia into the tetramic acid chloride 11. The pseudo acid chloride 3 was reacted further into the bis aziridine 8. Reduction of the mucochloric acid 1a furnished the trichlorofuranone 3. The cytotoxicity of these simple and bis‐cyclic butenolides have been evaluated in tissue culture on MAC13 and MAC16 cancer cell lines using the MTT cytotoxicity assay. The ester 5g, the acetate 4b and the carbamate 2b displayed a cytotoxicity in the low micromolar range. Further, an IC50 (50% inhibitory concentration) of 50 nM and 30 nM was determined for the epoxide 7 and the aziridine 8.

Antiviral Properties of the HIV-1 Proteinase Inhibitor RO 31-8959:

The peptide derivative Ro 31-8959 has been shown to be a potent inhibitor of HIV proteinase with an IC50 of 2 × 10−9M, against HIV-1RF in acutely infected lymphoblastoid cells. This inhibition was not overcome by increasing the infectious dose or by extending the culture time. Similar antiviral activity was also obtained against HIV-2, SIV and several AZT-resistant strains of HIV-1. The time of addition of the inhibitor could be delayed for 22 h without significant loss of activity, supporting its mode of action as taking place late in the replication cycle of HIV-1. Ro 31-8959 also showed activity against chronically infected cells.

Screening of Compounds for Activity against HIV: A Collaborative Study

The collaborative study was undertaken to examine the sensitivity of a range of tests used in assessing the antiviral activities of compounds against human immunodeficiency virus (HIV). A panel of 20 compounds with diverse antiviral activities against HIV were tested under code at three antiviral testing centres supported by the Medical Research Councils AIDS Directed Programme and at the European Community Centralised Facility (ECCF) for New Antiviral Compounds against AIDS in Belgium. Compounds known to have major anti-HIV activity ranked high in all assays, with the exception of the glucosidase inhibitors and certain nucleoside analogues. Results of two assays based on MT4 cells (centre IV) showed a high degree of similarity, despite the use of distinct HIV-1 (HTLV-IIIB) and HIV-2 (ROD) viruses. Considerable similarity was also observed between the assays based on HTLV-IIIRF in C8166 cells (centres I and II). Other assays performed at centre II and at centre III had enhanced sensitivity for glycosidase inhibitors. The differences in anti-HIV activity that were observed may be attributable to specific properties of the cell lines used and particular testing methodologies. The use of more than one type of assay is advisable in order not to miss compounds with low to moderate activity against HIV.

T Cell Costimulation by Derivatives of Benzoic Acid

A number of analogues of benzoic acid were evaluated in a T cell costimulation assay. One compound, the sodium salt of 2-chloro-5-nitrobenzoic acid (CNBA-Na) was chosen for further study and was found to be a potent costimulator of anti-CD3-induced proliferation of both H9 lymphoblastoid cells (P<0.001) and human peripheral blood mononuclear cells (P=0.001) in a dose-dependent manner. The costimulatory effect of CNBA-Na on CD3-triggered DNA synthesis did not enhance human immunodeficiency virus replication in infected cells. Studies with blocking monoclonal antibodies against B7-1 or B7-2 indicated that the immunopotentiatory effect of CNBA-Na required a macromolecular interaction between CD28 (a costimulatory receptor on T cells) and its counter receptor B7 expressed on antigen-presenting cells. The discovery that this low molecular weight compound causes T cell proliferation highlights a potentially novel therapeutic approach to immunodeficiency diseases.