We have demonstrated the anti-HIV-1 activity of four salicylidene acylhydrazides. These compounds inhibit diverse clade B HIV-1 strains in the low μM range, are active with primary CD4+ lymphoblast target cells and maintain their activity in the presence of vaginal fluid and semen simulant. Moreover, we have shown that HIV-1 inhibitory activity is reversible when high concentrations of Fe2+ are added to the medium. Finally, the compounds appear to inhibit infection at a post-integration stage.
An ideal vaginal microbicide would have inhibitory activity against a number of sexually transmitted pathogens. Notably, the four compounds we have tested inhibit three different R5 clinical isolates of HIV-1 and have similar inhibitory potency against C. trachomatis
]. Moreover, compound INP0341was also active against five of six N. gonorrhoeae
strains with minimal bactericidal concentrations of 6.3 μM [5
]. In the case of Chlamydia
, compound INP0341 has been shown to be active in a mouse model of vaginal infection [5
]. While inhibiting a number of pathogens, the compounds tested do not have inhibitory activity (at concentrations up to 100μM) against two of the most common hydrogen peroxide-producing members of the vaginal normal flora, Lactobacillus jensenii
and Lactobacillus crispatus
]. These two organisms are considered crucial to the maintenance of normal vaginal flora. Thus, the specificity of these compounds in targeting pathogens and sparing essential organisms make them well-suited for development as topical vaginal microbicides.
The four compounds had activity against HIV-1 in primary CD4+ lymphoblasts obtained from healthy donors. Since CD4+ lymphocytes are the major target cell for HIV-1 replication, compound activity could translate into in vivo efficacy. However, we have not measured the effect of the salicylidene acylhydrazides on other cells, such as macrophages and dendritic cells, which are likely very early targets following mucosal exposure. Moreover, although a vaginal fluid simulant at three different pH values, as well as semen simulant and a combination of semen and vaginal fluid simulants had little or no effect on activity, it remains to be determined if the compounds will inhibit HIV infection after exposure to intact or inflamed vaginal mucosa.
In the absence of details regarding the exact nature of the inoculum during natural sexual transmission of HIV, effective preventions should be active against incoming virus in both the cell-free and cell-associated forms. In this regard, the compounds tested were able to inhibit virus after exposure of target cells to either cell-free or cell- associated virus.
Our experiments clearly show that the addition of iron can reverse the inhibitory effect of the four compounds tested. Previous studies have also found that iron chelating agents, including the salicylidene acylhydrazide 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazine, inhibit HIV-1 [7
]. Moreover, increased iron stores associated with a number of conditions, adversely affect HIV infection [16
]. Our results are also consistent with those of others who have observed that iron depletion affects HIV replication at the level of transcription and that such an effect is related to perturbations in cell cycling [7
]. We have previously observed that the inhibition of C. trachomatis
and N. gonorrhoeae
by salicylidene acylhydrazides is also reversible by iron [4
]. Finally, a group of acylhydrazones has been shown to inhibit SIV, bind to HIV-1 capsid, and inhibit capsid assembly [19
]. Whether the compounds we tested also target capsid assembly or solely act as iron chelators is unknown.
By likely acting on a post-integration step in virus replication, the compounds tested will allow the entry of virus and the integration of its genome. This implies that, in order to be effective, the compounds will need to be maintained at inhibitory concentrations for the duration of the life of infected cells. In this regard, we are in the process of testing the compounds in a humanized mouse model to gauge there in vivo efficacy. Moreover, the mechanism of inhibition will likely require the use of these compounds in combination with substances that inhibit early steps in the virus life cycle.
The compounds used in our study have been shown to slow cell progression, but as demonstrated by the LDH and PI results, there is minimal toxicity to the cells at the active concentrations. We note that the TI values are rather small, particularly for INP0400. However, because of compound precipitation at high concentrations, TI’s were calculated using an unconventional 25% cytotoxicity value, rather than the usual 50% value. In addition, 1mM of INP0341 applied intravaginally to mice on three separate occasions over three days did not result in histological abnormalities [6
]. In a previous study, INP0161, 0341 and 0400 also showed minimal toxicity to HeLa 299 cells when measured by mitochondrial dehydrogenase activity; however, in that study, INP0149 resulted in 9.4% and 81.1% cytotoxicity at 20μM and 50μM [4
]. Additonal in vivo
studies will be required to further establish the safety profile of these compounds.
In summary, we have identified salicylidene acylhydrazides with in vitro
anti-HIV-1 activity in the μM range. Although not as potent as some anti-retroviral agents, these compounds appear to have relatively low cytotoxicity in vitro
and in vivo
at well above inhibitory concentrations [6
]. Finally, the activity of the compounds against other sexually transmitted pathogens makes them, likely in combination with other agents, potential candidates for formulation and use in a broad-spectrum topical microbicide.