In vitro anti-HIV-1 activity of salicylidene acylhydrazide compounds

Salicylidene acylhydrazide compounds have been shown to inhibit bacterial pathogens, including Chlamydia and Neisseria gonorrhoeae. If such compounds could also target HIV-1, their potential use as topical microbicides to prevent sexually transmitted infections would be considerable. In this study, the in vitro anti-HIV-1 activity, cytotoxicity and mechanism of action of several salicylidene acylhydrazides were determined. Inhibitory activity was assessed using TZM-bl cells and primary peripheral blood mononuclear cells (PBMCs) as targets for HIV-1 infection. Antiviral activity was measured against cell-free and cell-associated virus and in vaginal fluid and semen simulants. Since the antibacterial activity of salicylidene acylhydrazides is reversible by Fe(2+), the ability of Fe(2+) and other cations to reverse the anti-HIV-1 activity of the compounds was determined. Real-time PCR was also employed to determine the stage affected in the HIV-1 replication cycle. Four compounds with 50% inhibitory concentrations against HIV-1 of 1-7μM were identified. In vitro toxicity varied but was generally limited. Activity was similar against three R5 clade B primary isolates and whether the target for virus replication was TZM-bl cells or PBMCs. Compounds inhibited cell-free and cell-associated virus and were active in vaginal fluid and semen simulants. Fe(2+), but not other cations, reversed the anti-HIV-1 effect. Finally, the inhibitory effect of the compounds occurred at a post-integration step. In conclusion, salicylidene acylhydrazides were identified with in vitro anti-HIV-1 activity in the micromolar range. The activity of these compounds against other sexually transmitted pathogens makes them potential candidates to formulate for use as a broad-spectrum topical genital microbicide.     

Neurokinin-1 Receptor Antagonist (Aprepitant) Suppresses HIV-1 Infection of Microglia/Macrophages

Neurokinin-1 receptor (NK-1R) antagonists suppress HIV-1 infection of macrophages in vitro. We have further investigated the anti-HIV-1 activity of aprepitant, a Food and Drug Administration-approved NK-1R antagonist, and its cytotoxic effect in the macrophage/microglia system. Aprepitant inhibited infection of macrophages with primary HIV-1 R5 strains (subtypes A, D, and H; UG275, BZ163, and BCF-KITA), while it had little effect on primary HIV-1 X4 strains (subtypes B and D, BZ167 and SE365). Aprepitant, when added to microglia cultures infected with CSF-derived HIV-1 strains (JAGO or JRFL), significantly inhibited viral replication. Aprepitant also enhanced the anti-HIV-1 activity of enfuvirtide (an HIV-1 fusion inhibitor) in HIV-1-infected macrophages. Over a concentration range of 10⁻⁹ to 10^–5 M, aprepitant had little cytotoxic effect (less than 10%) on macrophages during the in vitro cultures. Autologous human serum (≤20%) had little effect on the anti-HIV-1 activity of aprepitant in macrophages. These observations provide additional evidence to support the potential use of NK-1R antagonists as therapeutic and immunomodulatory agents for the treatment of HIV-1 infection.     

Crystal structure of tert-butyldimethylsilyl-spiroaminooxathioledioxide-thymine (TSAO-T) in complex with HIV-1 reverse transcriptase (RT) redefines the elastic limits of the non-nucleoside inhibitor-binding pocket

tert-Butyldimethylsilyl-spiroaminooxathioledioxide (TSAO) compounds have an embedded thymidine-analogue backbone; however, TSAO compounds invoke non-nucleoside RT inhibitor (NNRTI) resistance mutations. Our crystal structure of RT:7 (TSAO-T) complex shows that 7 binds inside the NNRTI-binding pocket, assuming a "dragon" shape, and interacts extensively with almost all the pocket residues. The structure also explains the structure-activity relationships and resistance data for TSAO compounds. The binding of 7 causes hyper-expansion of the pocket and significant rearrangement of RT subdomains. This nonoptimal complex formation is apparently responsible (1) for the lower stability of a RT (p66/p51) dimer and (2) for the lower potency of 7 despite of its extensive interactions with RT. However, the HIV-1 RT:7 structure reveals novel design features such as (1) interactions with the conserved Tyr183 from the YMDD-motif and (2) a possible way for an NNRTI to reach the polymerase active site that may be exploited in designing new NNRTIs.     

Anti-HIV activity of dextran sulphate as determined under different experimental conditions

Dextran sulphate is a potent and selective inhibitor of human immunodeficiency virus type 1 (HIV-1). Its anti-HIV-1 activity has been investigated under varying experimental conditions. MT-4 cells were infected with HIV-1 at different multiplicities of infection (MOI), and treated with either dextran sulphate, 3'-azido-2',3'-dideoxythymidine (AZT), or anti-HIV-1 serum obtained from an ARC patient. Dextran sulphate suppressed HIV-1 replication (as monitored by viral antigen expression) when the MOI was 0.01 or 0.1. It was ineffective at an MOI of 1.0. The anti-HIV-1 serum was only partially effective at an MOI of 0.01 and ineffective at an MOI of 0.1 or 1.0. AZT proved effective at all three MOIs. Co-cultures of uninfected and HIV-1-infected MT-4 cells were protected against destruction by dextran sulphate at a concentration of 10 and 100 micrograms/ml. To fully suppress viral antigen expression a concentration of 100 micrograms/ml was needed. When used at this concentration, a 1-h contact of dextran sulphate with the cells during the virus adsorption period sufficed to suppress HIV-1 antigen expression. In this sense, dextran sulphate behaved like the anti-HIV-1 serum. Dextran sulphate also behaved like OKT-4A in that they both inhibited HIV-1 attachment to the MT-4 cells, whereas OKT-4 failed to do so. However, dextran sulphate did not affect the binding of OKT-4A to the cells. The present results support the concept that dextran sulphate owes its anti-HIV-1 activity mainly to inhibition of virus binding to its target cells. The anti-HIV-1 activity of dextran sulphate is highly dependent on its sulphate content.     

白藜芦醇衍生物体外抗HIV-1活性的初步研究

目的:检测白藜芦醇衍生物IFB-1和IFB-9的体外抗HIV-1活性,并对其进行抗HIV-1作用机制的初步研究。方法:采用MTT比色法检测白藜芦醇衍生物IFB-1和IFB-9的细胞毒性;细胞病变法检测化合物对HIV-1急性感染的抑制活性;采用HIV-1 p24抗原ELISA方法检测临床分离株HIV-1KM018在PBMC中复制的抑制实验;采用细胞病变法检测HIV-1感染和未感染细胞之间的融合;采用HIV-1重组逆转录酶活性抑制实验,HIV-1重组蛋白酶活性抑制实验以及直接杀病毒实验来研究化合物体外抗HIV-1机制。结果:白藜芦醇衍生物IFB-1和IFB-9对HIV-1IIIB诱导的合胞体形成抑制的选择指数分别为16.16和230.27;IFB-1和IFB-9均能抑制p24抗原的产生,EC50s分别为14.51和0.23μg.mL-1,也能抑制HIV-1KM018在PBMC中的复制。IFB-1和IFB-9对感染细胞与未感染细胞融合有较好的抑制,但对病毒逆转录酶和蛋白酶体外活性没有抑制作用,也不能直接杀死HIV-1病毒。结论:白藜芦醇衍生物IFB-1和IFB-9具有抗HIV-1活性,其作用机制可能为抑制病毒进入细胞。     

Design of MKC-442 (emivirine) analogues with improved activity against drug-resistant HIV mutants

Two analogues of the nonnucleoside inhibitor of HIV-1 RT, MKC-442 (emivirine), containing different C6 substituents have been designed to be less susceptible to the commonly found drug-resistance mutation of Tyr181Cys. Compound TNK-6123 had a C6 thiocyclohexyl group designed to have more flexibility in adapting to the mutated drug-binding site. GCA-186 had additional 3',5'-dimethyl substituents aimed at forming close contacts with the conserved residue Trp229. Both compounds showed approximately 30-fold greater inhibitory effect than MKC-442 to the Tyr181Cys mutant virus as well as to the clinically important Lys103Asn virus. X-ray crystallographic structure determination of complexes with HIV-1 RT confirmed the predicted binding modes. These strategies might be used to improve the resilience of other NNRTI series against common drug-resistance mutations.