Conserved hydrogen bonds and water molecules in MDR HIV-1 protease substrate complexes

The success of highly active antiretroviral therapy (HAART) in anti-HIV therapy is severely compromised by the rapidly developing drug resistance. HIV-1 protease inhibitors, part of HAART, are losing their potency and efficacy in inhibiting the target. Multi-drug resistant (MDR) 769 HIV-1 protease (resistant mutations at residues 10, 36, 46, 54, 62, 63, 71, 82, 84, 90) was selected for the present study to understand the binding to its natural substrates. The nine crystal structures of MDR769 HIV-1 protease substrate hepta-peptide complexes were analyzed in order to reveal the conserved structural elements for the purpose of drug design against MDR HIV-1 protease. Our structural studies demonstrated that highly conserved hydrogen bonds between the protease and substrate peptides, together with the conserved crystallographic water molecules, played a crucial role in the substrate recognition, substrate stabilization and protease stabilization. In addition, the absence of the key flap-ligand bridging water molecule might imply a different catalytic mechanism of MDR769 HIV-1 protease compared to that of wild type (WT) HIV-1 protease.     

The human immunodeficiency virus (HIV) protease inhibitor indinavir directly affects the opportunistic fungal pathogen Cryptococcus neoformans

Highly active antiretroviral therapy (HAART), that includes human immunodeficiency virus (HIV) protease inhibitors (PIs), has been remarkably efficacious including against some opportunistic infections. In this report we investigated the effect(s) of the PI indinavir on protease activity by Cryptococcus neoformans, an opportunistic fungal pathogen responsible for recurrent meningoencephalitis in AIDS patients. Indinavir was also tested for potential effects on other parameters, such as fungal viability, growth ability and susceptibility to immune effector cells. It was found that indinavir impaired cryptococcal protease activity in a time- and dose-dependent fashion. The phenomenon was similarly detectable in ATCC/laboratory strains and clinical isolates. C. neoformans growth rate was also significantly reduced upon exposure to indinavir, while fungal viability was not affected and mitochondrial toxicity not detected. Furthermore, as assessed by an in vitro infection model, indinavir significantly and consistently augmented C. neoformans susceptibility to microglial cell-mediated phagocytosis and killing. Overall, by providing the first evidence that indinavir directly affects C. neoformans, these data add new in vitro insights on the wide-spectrum efficacy of PIs, further arguing for the clinical relevance of HAART against opportunistic infections in AIDS.     

Structure of an Fab-protease complex reveals a highly specific non-canonical mechanism of inhibition

The vast majority of protein protease inhibitors bind their targets in a substrate-like manner. This is a robust and efficient mechanism of inhibition but, due to the highly conserved architecture of protease active sites, these inhibitors often exhibit promiscuity. Inhibitors that show strict specificity for one protease usually achieve this selectivity by combining substrate-like binding in the active site with exosite binding on the protease surface. The development of new, specific inhibitors can be aided greatly by binding to non-conserved regions of proteases if potency can be maintained. Due to their ability to bind specifically to nearly any antigen, antibodies provide an excellent scaffold for creating inhibitors targeted to a single member of a family of highly homologous enzymes. The 2.2 Å resolution crystal structure of an Fab antibody inhibitor in complex with the serine protease membrane-type serine protease 1 (MT-SP1/matriptase) reveals the molecular basis of its picomolar potency and specificity. The inhibitor has a distinct mechanism of inhibition; it gains potency and specificity through interactions with the protease surface loops, and inhibits by binding in the active site in a catalytically non-competent manner. In contrast to most naturally occurring protease inhibitors, which have diverse structures but converge to a similar inhibitory archetype, antibody inhibitors provide an opportunity to develop divergent mechanisms of inhibition from a single scaffold.     

HIV-1 dynamics at different time scales under antiretroviral therapy

We exploit a model that considers three compartments: blood plasma (BP), lymphoid tissue-interstitial spaces (LT-IS), and follicular dendritic cells (FDC), for the HIV-1 dynamics under the application of highly active antiretroviral therapy (HAART) which allowed us to unravel distinct viral dynamics occurring in short- (2 days), middle- (21 days), and long-term (183 days) time scales. The different time scales are determined by the viral clearance rate, the ratio of productively infected CD4⁺ T cells to chronically infected cells, and the dissociation rate of HIV-1 complexes from FDC. This generates a scenario in which, after an initial transient stage, the viral BP dynamics decouples and becomes governed by the lymphoid tissue (LT) dynamics; in a later stage, a new decoupling occurs in which the LT-IS dynamics is slaved to that of the FDC dynamics. We observed an initial increase in the viremia after HAART in a patient who did not receive protease inhibitors (PI). By means of the above-mentioned model we were able to highlight the relevant parameters which need to be estimated at three different time scales after HAART.     

In vitro evidence of inhibition of mitochondrial protease processing by HIV-1 protease inhibitors in yeast: a possible contribution to lipodystrophy syndrome

Highly active antiretroviral therapy has been associated with the emergence of lipodystrophy syndromes that have clinical features commonly seen in patients with mitochondrial dysfunction. The effect of therapeutic protease inhibitors (PIs) on mitochondrial function is unknown. Mitochondrial matrix space proteins possess an amino-terminal leader peptide that is removed by the mitochondrial processing protease (MPP). Lack of cleavage could result in non- or dysfunctional mitochondrial proteins. The effects of different PIs on protease processing using pure MPP or yeast mitochondria, recognized models for mammalian counterparts, were examined in vitro. Multiple PIs were found to inhibit MPP, evidenced by accumulation of immature pALDH and decreased levels of processed ALDH. Both indinavir and amprenavir at 5.0 mg/ml resulted in significant inhibition of MPP. Although inhibition of MPP was also observed with ritonavir and saquinavir, the inhibition was difficult to quantify due to background inhibition of MPP by DMSO that was required to solubilize the drugs for the in vitro studies. Indinavir was also shown to inhibit MPP within yeast mitochondria. Lack of processing may impair mitochondrial function and contribute to the observed mitochondrial dysfunctions in patients receiving HAART and implicated in antiretroviral-associated lipodystrophy.     

Highly Active AntiRetroviral Therapy and opportunistic protozoan infections

Opportunistic parasite infections (OPIs) are an important cause of morbidity and mortality in persons infected with HIV. In industrialised countries, the use of Highly Active AntiRetroviral Therapy (HAART) results to be effective in suppressing the HIV viral load, with a quantitative and qualitative improvement in the CD4+ T-cell count followed by a strong reduction of opportunistic infections including those caused by parasites. These successes have been mainly attributed to the reconstitution of the cell immunity, which play the most important role in controlling OPIs. However, there are many clinical reports and several laboratory results, which suggest that the control of OPIs in HIV-positive persons under HAART is also induced by the anti-HIV protease inhibitors (PIs), which inhibit the aspartyl proteases of the parasites. The non-conventional use of HIV-PIs seems to be an alternative way for the treatment of parasitic infections, which should be deeply investigated. Of five longitudinal studies carried out before and after the introduction of HAART, four studies showed a strong reduction of toxoplasmic encephalitis (TE) in HIV-positive persons under HAART, whereas in another study, no difference was observed in the incidence rate of TE before and after the introduction of HAART. The influence of HAART in reducing TE has been also confirmed in a randomised, controlled clinical trial, which showed that there is no increase in the risk of developing TE after beginning HAART, even though HIV-infected persons with TE had a discontinuing prophylaxis for Toxoplasma gondii. Four HIV protease inhibitors were tested against the T. gondii virulent RH strain in vitro, alone or in association with pyrimethamine or sulfadiazine. Ritonavir and nelfinavir were highly inhibitory for the parasite growth. Furthermore, none of the antiviral drugs negatively affected the anti-Toxoplasma activity of pyrimethamine or sulfadiazine. In HIV-Leishmania co-infections, a changing pattern has been observed in the HAART era, characterised by a high rate of relapses, which could be explained by the increased survival rate resulting from the effective antiretroviral therapy. A 64.8% decrease of the visceral leishmaniasis incidence was detected after HAART began to be used extensively in Spain. In a large cohort study carried out in ten European countries and in Australia, the relative risk to contract cryptosporidiosis as the first AIDS defining disease was reduced by 96% in the HAART era. In Italy, the relative risk of death for cryptosporidiosis reduced of 74% in the period 1997-98, when HIV-positive persons received HAART. In a large study carried out in Italy, isosporiasis was included in the group of opportunistic infections, of which the relative hazards showed a reduction of 95% in the HAART era. Since 1997, there was the evidence that the use of HAART in persons with advanced HIV infection can improve chronic diarrhoea and lead to disappearance of Enterocytozoon bieneusi from the stools. Although the reconstitution of the cellular immunity seems to be the main factor influencing the reduction of OPIs in persons with AIDS who undergo HAART, there are clinical and microbiological evidences, as well as in vitro and in vivo results, which indicate direct effects of HIV-PIs on the proteases of opportunistic parasites. These findings stress the existence of non-conventional unexpected benefits of PIs in HAART against protozoa. In addition, this benefit of PIs has been demonstrated also for Candida albicans secreted aspartyl proteases and for P. carinii acid proteases. In spite of these important results, HIV PIs are still very toxic for humans, specially in cases of very long treatment, and no clinical trial has been carried out for persons at risk, such as children and pregnant women, because the priority was to reduce the severity of HIV and not the evaluation of possible side effects of the therapy. It follows that further researches are needed to establish the non-conventional use of HIV PIs. Furthermore, the study of PIs against specific aspartyl proteases of those opportunistic protozoa that cause severe and intractable diseases, could be considered an alternative way towards the development of new drugs that may prove effective against these infections.     

Cysteine protease inhibitors reduce brain beta-amyloid and beta-secretase activity in vivo and are potential Alzheimer's disease therapeutics

Beta-secretase inhibitors that lower brain beta-amyloid peptides (Abeta) are likely to be effective for treating Alzheimer's disease (AD). Irreversible epoxysuccinyl cysteine protease inhibitors are known to reduce brain Abeta and beta-secretase activity in the guinea pig model of human Abeta production. In this study, acetyl-L-leucyl-L-valyl-L-lysinal (Ac-LVK-CHO) is also shown to significantly reduce brain Abeta and beta-secretase activity and brain Abeta in the same model. Ac-LVK-CHO is structurally distinct from the epoxysuccinyl inhibitors and is a reversible cysteine protease inhibitor. The results suggest that cysteine protease inhibitors generally, and reversible cysteine protease inhibitors specifically, have potential for development as AD therapeutics.