Pascal Clayette

Porous metal-organic-framework nanoscale carriers as a potential platform for drug delivery and imaging

In the domain of health, one important challenge is the efficient delivery of drugs in the body using non-toxic nanocarriers. Most of the existing carrier materials show poor drug loading (usually less than 5 wt% of the transported drug versus the carrier material) and/or rapid release of the proportion of the drug that is simply adsorbed (or anchored) at the external surface of the nanocarrier. In this context, porous hybrid solids, with the ability to tune their structures and porosities for better drug interactions and high loadings, are well suited to serve as nanocarriers for delivery and imaging applications. Here we show that specific non-toxic porous iron(III)-based metal-organic frameworks with engineered cores and surfaces, as well as imaging properties, function as superior nanocarriers for efficient controlled delivery of challenging antitumoural and retroviral drugs (that is, busulfan, azidothymidine triphosphate, doxorubicin or cidofovir) against cancer and AIDS. In addition to their high loadings, they also potentially associate therapeutics and diagnostics, thus opening the way for theranostics, or personalized patient treatments.

Regulated Expression of Sodium-dependent Glutamate Transporters and Synthetase: a Neuroprotective Role for Activated Microglia and Macrophages in HIV Infection?

It is now widely accepted that neuronal damage in HIV infection results mainly from microglial activation and involves apoptosis, oxidative stress and glutamate‐mediated neurotoxicity. Glutamate toxicity acts via 2 distinct pathways: an excitotoxic one in which glutamate receptors are hyperactivated, and an oxidative one in which cystine uptake is inhibited, resulting in glutathione depletion and oxidative stress. A number of studies show that astrocytes normally take up glutamate, keeping extracellular glutamate concentration low in the brain and preventing excitotoxicity. This action is inhibited in HIV infection, probably due to the effects of inflammatory mediators and viral proteins. Other in vitro studies as well as in vivo experiments in rodents following mechanical stimulation, show that activated microglia and brain macrophages express high affinity glutamate transporters. These data have been confirmed in chronic inflammation of the brain, particularly in SIV infection, where activated microglia and brain macrophages also express glu‐tamine synthetase. Recent studies in humans with HIV infection show that activated microglia and brain macrophages express the glutamate transporter EAAT‐1 and that expression varies according to the disease stage. This suggests that, besides their recognized neurotoxic properties in HIV infection, these cells also have a neuroprotective function, and may partly make up for the inhibited astrocytic function, at least temporarily. This hypothesis might explain the discrepancy between microglial activation which occurs early in the disease, and neuronal apoptosis and neuronal loss which is a late event. In this review article, we discuss the possible neuro‐protective and neurotrophic roles of activated microglia and macrophages that may be generated by the expression of high affinity glutamate transporters and glutamine synthetase, 2 major effectors of glial glutamate metabolism, and the implications for HIV‐induced neuronal dysfunction, the underlying cause of HIV dementia.

Towards an Improved anti-HIV Activity of NRTI via Metal–Organic Frameworks Nanoparticles

Nanoscale mesoporous iron carboxylates metal-organic frameworks (nanoMOFs) have recently emerged as promising platforms for drug delivery, showing biodegradability, biocompatibility and important loading capability of challenging highly water-soluble drugs such as azidothymidine tryphosphate (AZT-TP). In this study, nanoMOFs made of iron trimesate (MIL-100) were able to act as efficient molecular sponges, quickly adsorbing up to 24 wt% AZT-TP with entrapment efficiencies close to 100%, without perturbation of the supramolecular crystalline organization. These data are in agreement with molecular modelling predictions, indicating maximal loadings of 33 wt% and preferential location of the drug in the large cages. Spectrophotometry, isothermal titration calorimetry, and solid state NMR investigations enable to gain insight on the mechanism of interaction of AZT and AZT-TP with the nanoMOFs, pointing out the crucial role of phosphates strongly coordinating with the unsaturated iron(III) sites. Finally, contrarily to the free AZT-TP, the loaded nanoparticles efficiently penetrate and release their cargo of active triphosphorylated AZT inside major HIV target cells, efficiently protecting against HIV infection.

Anti-Human Immunodeficiency Virus Activity of Tau Interferon in Human Macrophages: Involvement of Cellular Factors and β-Chemokines

ABSTRACT Tau interferon (IFN-τ) is a noncytotoxic type I IFN responsible for maternal recognition of the fetus in ruminants. IFN-τ inhibits human immunodeficiency virus (HIV) replication more strongly than human IFN-α, particularly in human monocyte-derived macrophages. In this study performed in human macrophages, IFN-τ efficiently inhibited the early steps of the biological cycle of HIV, decreasing intracellular HIV RNA and inhibiting the initiation of the reverse transcription of viral RNA into proviral DNA. Two mechanisms induced by IFN-τ treatment in macrophages may account for this inhibition: (i) the synthesis of the cellular antiviral factors such as 2′,5′-oligoadenylate synthetase/RNase L and MxA protein and (ii) an increased production of MIP-1α, MIP-1β, and RANTES, which are natural ligands of CCR5, the principal coreceptor of HIV on macrophages. Our results suggest that IFN-τ induces the same antiviral pathways in macrophages as other type I IFNs but without associated toxicity.

Inhibition of HIV-1 Replication by Cell-penetrating Peptides Binding Rev

New therapeutic agents able to block HIV-1 replication are eagerly sought after to increase the possibilities of treatment of resistant viral strains. In this report, we describe a rational strategy to identify small peptide sequences owning the dual property of penetrating within lymphocytes and of binding to a protein target. Such sequences were identified for two important HIV-1 regulatory proteins, Tat and Rev. Their association to a stabilizing domain consisting of human small ubiquitin-related modifier-1 (SUMO-1) allowed the generation of small proteins named SUMO-1 heptapeptide protein transduction domain for binding Tat (SHPT) and SUMO-1 heptapeptide protein transduction domain for binding Rev (SHPR), which are stable and efficiently penetrate within primary lymphocytes. Analysis of the antiviral activity of these proteins showed that one SHPR is active in both primary lymphocytes and macrophages, whereas one SHPT is active only in the latter cells. These proteins may represent prototypes of new therapeutic agents targeting the crucial functions exerted by both viral regulatory factors.

Differential Expression Levels of MRP1, MRP4, and MRP5 in Response to Human Immunodeficiency Virus Infection in Human Macrophages

ABSTRACT Multidrug resistance proteins (MRPs) have been reported to be involved in the efflux of some anti-human immunodeficiency virus (HIV) drugs. We show here that MRP1, MRP4, and MRP5 are expressed at the mRNA level in human monocyte-derived macrophages. HIV infection caused increased transcription of these MRPs; however, temporal differences in stimulation are reported.

Long-Lasting Enfuvirtide Carrier Pentasaccharide Conjugates with Potent Anti-Human Immunodeficiency Virus Type 1 Activity

ABSTRACT Enfuvirtide (also known as Fuzeon, T-20, or DP-178) is an antiretroviral fusion inhibitor which prevents human immunodeficiency virus type 1 (HIV-1) from entering host cells. This linear 36-mer synthetic peptide is indicated, in combination with other antiretroviral agents, for the treatment of HIV-1-infected individuals and AIDS patients with multidrug-resistant HIV infections. Although enfuvirtide is an efficient anti-HIV-1 drug, its clinical use is limited by a short plasma half-life, i.e., approximately 2 h, which requires twice-daily subcutaneous injections, often resulting in skin sensitivity reaction side effects at the injection sites. Ultimately, 80% of patients stop enfuvirtide treatment within 6 months because of these side effects. We report on the development of long-lasting enfuvirtide conjugates by the use of the site-specific conjugation of enfuvirtide to an antithrombin-binding carrier pentasaccharide (CP) through polyethylene glycol (PEG) linkers of various lengths. These conjugates showed consistent and broad anti-HIV-1 activity in the nanomolar range. The coupling of the CP to enfuvirtide only moderately affected the in vitro anti-HIV-1 activity in the presence of antithrombin. Most importantly, one of these conjugates, enfuvirtide-PEG12-CP (EP40111), exhibited a prolonged elimination half-life of more than 10 h in rat plasma compared to the half-life of native enfuvirtide, which was 2.8 h. On the basis of the pharmacokinetic properties of antithrombin-binding pentasaccharides, the anticipated half-life of EP40111 in humans would putatively be about 120 h, which would allow subcutaneous injection once a week instead of twice daily. In conclusion, EP40111 is a promising compound with strong potency as a novel long-lasting anti-HIV-1 drug.

A New Generation of Peptide-based Inhibitors Targeting HIV-1 Reverse Transcriptase Conformational Flexibility

The biologically active form of human immunodeficiency virus (HIV) type 1 reverse transcriptase (RT) is a heterodimer. The formation of RT is a two-step mechanism, including a rapid protein-protein interaction “the dimerization step,” followed by conformational changes “the maturation step,” yielding the biologically active form of the enzyme. We have previously proposed that the heterodimeric organization of RT constitutes an interesting target for the design of new inhibitors. Here, we propose a new class of RT inhibitors that targets protein-protein interactions and conformational changes involved in the maturation of heterodimeric reverse transcriptase. Based on a screen of peptides derived from the thumb domain of this enzyme, we have identified a short peptide PAW that inhibits the maturation step and blocks viral replication at subnanomolar concentrations. PAW only binds dimeric RT and stabilizes it in an inactive/non-processive conformation. From a mechanistic point of view, PAW prevents proper binding of primer/template by affecting the structural dynamics of the thumb/fingers of p66 subunit. Taken together, these results demonstrate that HIV-1 RT maturation constitutes an attractive target for AIDS chemotherapeutics.

PMS-601, a New Platelet-Activating Factor Receptor Antagonist That Inhibits Human Immunodeficiency Virus Replication and Potentiates Zidovudine Activity in Macrophages

ABSTRACT We assessed the anti-human immunodeficiency virus (anti-HIV) activity in vitro of new platelet-activating factor (PAF) receptor antagonists, as PAF and viral replication are thought to be involved in HIV neuropathogenesis. We found that PMS-601 inhibited proinflammatory cytokine synthesis and HIV replication in macrophages and potentiated the antiretroviral activity of zidovudine. These results suggest that PMS-601 is of potential value as an adjuvant treatment for HIV infection.

Infection of human macrophages with an endogenous tumour necrosis factor-α (TNF-α)-independent human immunodeficiency virus type 1 isolate is unresponsive to the TNF-α synthesis inhibitor RP 55778

Monocyte-derived macrophages (MDM) were demonstrated to be susceptible to productive infection by the monocytotropic human immunodeficiency virus type 1 (HIV-1) strain HIV-1/Ba-L and by three primary HIV-1 isolates, HIV-1/DAS, HIV-1/PAR and HIV-1/THI. Production of tumour necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and IL-1 beta was monitored between days 3 and 26 after MDM infection. TNF-alpha and IL-6 were detected in cell culture supernatants from days 16 to 21 following HIV-1/DAS, HIV-1/PAR and HIV-1/Ba-L infection, at the time of high viral replication. IL-1 beta was not found at the same time points. TNF-alpha mRNA expression occurred around the peak of both TNF-alpha levels and supernatant RT activities. In HIV-1/THI-infected macrophage cultures no endogenously produced TNF-alpha was observed, despite high levels of HIV-1 in MDM. This result demonstrates that a primary isolate may replicate independently of TNF-alpha in MDM. To investigate the relationship between TNF-alpha and viral replication we used a TNF-alpha synthesis inhibitor, RP 55778. Treatment throughout the course of cell culture resulted in a significant decrease in both TNF-alpha levels and viral production in HIV-1/DAS-, HIV-1/PAR- and HIV-1/Ba-L-infected MDM cultures. This phenomenon is reversed by adding recombinant human TNF-alpha to the RP 55778-treated cell cultures from day 14 post-infection. No effect of RP 55778 was observed in MDM cultures infected with the primary isolate HIV-1/THI, whose replication is independent of TNF-alpha production and therefore remained unchanged after RP 55778 treatment. We conclude that the clinical value of such a drug is directly dependent on the ability of the HIV-1 strains involved to induce TNF-alpha production at the time of viral replication.