Photo Credit: C. Goldsmith, CDC The first treatments for HIV-1/AIDS targeted the viral enzymes, reverse transcriptase and protease; more recently, compounds that block viral fusion/entry have also been developed. HIV-1 regulatory proteins are considered to be more difficult drug targets, not least because ways to assay for activity are less clear, but researchers at the University of Pittsburgh have now described an assay for HIV-1 Nef and have identified compounds that block the function of this viral protein. The HIV-1 nef gene was originally believed to inhibit transcription and to be of little importance (hence ‘Negative regulatory Factor’), but the protein is now known to play key roles in viral replication and pathogenesis. Nef is one of the first proteins to be detected after infection of the host cell and has three main functions: it increases viral infectivity, down-regulates surface antigens, and alters signalling pathways to enhance survival of infected T-cells.
Nef binds to src family kinases via their SH3 domains and the Pittsburgh team have exploited Nef’s interaction with Hck (hemopoietic cell kinase) to develop an assay system suitable for high-throughput screening. In the assay, Hck activation is coupled to Nef, providing a direct readout of Nef activity. The assay was used to screen a library of 10,000 compounds biased towards kinase and phosphatase inhibitors, but also containing more diverse structures. The 4-amino substituted diphenylfuropyrimidine, DFP-4AP, which is structurally related to a series of recently described protein-tyrosine kinase inhibitors, was found to be active both in the initial FRET-based assay and in a cell-based assay using U87MG astroglioma cells engineered to express the HIV-1 receptors CD4 and CXCR4. DFP-4AP blocked wild-type HIV replication in U87MG cells with an IC50 value in the low µM range, but had no effect on replication of an HIV-ΔNef mutant, even at 10 µM, indicating a requirement for Nef in the antiviral activity of the compound.
The study, which is published in ACS Chemical Biology, establishes that coupling of Nef to one of its known host cell targets provides a viable high-throughput screen which can be used to identify small molecule inhibitors. Including Hck in the assay may also induce relevant conformations of both Hck and Nef that are essential for small molecule inhibitor binding and function, an idea that is supported by the enhanced potency and efficacy of the inhibitors in the kinase assay when Nef is present. It may be possible to use a similar coupled protein approach to identify compounds that block the function of other HIV virulence factors and compounds which inhibit the function of Nef – or other virulence factors – could eventually become new weapons in the fight against HIV/AIDS.
Although highly active antiretroviral therapy (HAART) has proved effective at controlling HIV-1 replication, eradication of the virus from an infected individual cannot be achieved with current treatments. Latently infected, resting memory CD4+ T-cells and macrophages act as reservoirs for the virus and so called “shock and kill” strategies (activation of HIV-1 in the presence of HAART to block viral spread followed by stimuli leading to cell death) have been proposed to eradicate the virus. Histone deacetylase inhibitors (HDACIs) have been shown to be effective for the “shock” phase, but their use is limited by toxicity at the levels needed for viral reactivation. A team of Italian researchers looked for ways to improve the effectiveness of HDACIs and found that adding buthionine sulfoximine (BSO) to class I HDACIs reduced the concentration of HDACI needed to reactivate HIV-1 in cell culture experiments. The team hypothesised that, since HIV-1 transcription is enhanced by oxidative stress, glutathione depletion might create an intracellular environment that facilitates HIV-1 activation by HDACIs. BSO inhibits γ-glutamylcysteine synthetase, the rate limiting enzyme in glutathione synthesis, and has been studied as an adjunct to chemotherapy. At non-toxic concentrations, class I HDACIs only reactivated virus in some of the cells but, when BSO was added, all of the cells responded and were killed. MS-275, a class I HDACI currently undergoing clinical trials for the treatment of cancer, was one of the compounds found to reactivate HIV-1 more effectively in the presence of BSO. The combination showed low toxicity in uninfected cells.
Although much more work needs to be done, the authors hope that their research may open up new avenues in the search for ways to completely eliminate HIV-1-infected cells from the body.
Concert Pharmaceuticals and GlaxoSmithKline recently announced a collaboration to develop deuterium-containing medicines, including CTP-518, a partially deuterated version of the HIV protease inhibitor, atazanavir (Reyataz™), marketed by Bristol-Myers Squibb. Reyataz™ is used in combination therapy to treat HIV/AIDS and, for most patients, the recommended dose is one 300mg tablet daily taken with ritonavir (Norvir™). Ritonavir was originally developed as a ‘stand-alone’ HIV protease inhibitor but is now primarily used, not for its antiviral activity, but to ‘boost’ levels of other protease inhibitors by inhibiting their metabolism. Despite its marked benefits as part of combination therapy, ritonavir is poorly tolerated by some patients and also influences the metabolism of concurrently administered drugs, especially those metabolised by CYP 3A4.
Concert is pioneering the modification of existing medicines by selectively replacing hydrogen atoms with deuterium atoms in the expectation that the modified compounds will have similar activity at the target enzyme or receptor, together with improved ADME properties. CTP-518 has been shown to have similar antiviral potency to atazanavir but slower hepatic metabolism, leading to the hope that it could be used clinically without the need for ‘boosting’ by ritonavir. This could lead to better safety and tolerability for patients and also allow for the inclusion of CTP-518 in fixed dose regimens. CTP-518 is expected to enter Phase I clinical trials in the second half of 2009.
Concert has filed a patent application (WO20081566632) claiming derivatives of atazanavir, including compounds 120 and 122.
The antiviral activities of compounds 120 and 122 were shown to be similar to, or slightly better than, that of atazanavir, both in the presence and absence of serum proteins. In human liver microsomes, compounds 120 and 122 showed an approximately 50% increase in half life compared with atazanavir. Following oral co-dosing in rats, compound 122 showed a 43% increase in half life, a 67% increase in Cmax and an 81% increase in AUC compared with atazanavir. When administered to chimps, both compounds showed around 50% increases in half life compared with atazanavir and about 2-fold increases in urine concentration.
In 1984, shortly after the confirmation of HIV as the causative agent of AIDS, the then United States Health and Human Services Secretary declared that a vaccine would be available within two years. Despite intensive efforts in many laboratories around the world, more than two decades later this ambition has still not been fulfilled.
In 2007, the unexpected and unequivocal failure of one of the largest clinic trials of a vaccine against HIV caused widespread disappointment and dismay but, writing in the journal Nature Medicine, researchers in the United States have now described a vaccination strategy that protects monkeys against infection with the simian immunodeficiency virus (SIV). Instead of trying to stimulate a response from the immune system as with previous vaccines, the new approach delivers genes for antibody-like proteins. The research team first created molecules known as immunoadhesins which bind to SIV and prevent it from infecting cells. After showing that these were effective in cell culture experiments, DNA encoding the SIV-specific immunoadhesins was injected into the monkeys’ muscles using an adeno-associated virus carrier system. The DNA then began to produce immunoadhesins that entered the blood circulation and protected the monkeys from infection. One month after treatment, the majority of the immunised monkeys were protected from infection following challenge with live SIV and all were protected against AIDS. In contrast, unimmunised monkeys all became infected and two thirds died from AIDS complications. High concentrations of the SIV-specific immunoadhesins remained in the immunised monkeys’ blood for over a year. The team hope that long-lasting and complete protection against HIV might also be achieved in humans and are planning initial clinical studies using neutralising antibodies based on those produced by the handful of people who have remained healthy despite long term infection with HIV.
Despite enormous efforts worldwide, there is still no effective way to prevent transmission of HIV-1. Some non-human primates produce θ-defensins (retrocyclins), which act as HIV-1 entry inhibitors by inhibiting fusion of HIV-1 Env. In humans, however, θ-defensin genes have a premature stop codon that blocks translation. Defensins are small cationic, tri-disulfide bonded peptides expressed by leukocytes and epithelial cells that contribute to immune defenses against bacterial and viral infections. In primates, there are three sub-families of defensins, α-, β-, and θ-, classified on the basis of cysteine position and disulphide bonding pattern. In rhesus macaques, formation of θ-defensins, which are 18-residue cyclic peptides and the only known cyclic peptides in mammals, involves post-translational head-to-tail ligation of two nonapeptides. It was not known whether this process could occur in human cells, but researchers at the University of Central Florida, working with scientists from the University of California and the Centers for Disease Control, have now demonstrated that human cells retain the ability to produce retrocyclins and have further used the aminoglycoside antibiotics, gentamicin and tobramycin, to read-through the premature stop codon found in the mRNA transcripts and induce synthesis of bioactive retrocyclins. Although much more work needs to be done to demonstate the safety and effectiveness of this approach, the study provides hope that topical aminoglycoside-based microbicides – or other compounds that allow read-through of the stop codon –could one day be used topically to prevent sexual transmission of HIV-1.
Topical application of viral entry inhibitors or other microbicides is an attractive strategy to prevent sexual transmission of the human immunodeficiency virus (HIV). Griffithsin, a protein isolated from the red algae Griffithsia sp which grows off the coast of New Zealand, has been shown in vitro to be a potent HIV entry inhibitor, but the cost of production has so far hampered its development.
Writing in the March 30th Early Edition of PNAS, a multinational team of scientists has now described a breakthrough which should allow the manufacture and isolation of significant amounts of griffithsin as an agricultural crop. Griffithsin was shown to accumulate to a level of more than 1 gram of recombinant protein per kilogram of leaf material of Nicotiana benthamiana when expressed via an infectious tobacco mosaic virus vector. Nicotiana benthamiana, which is native to Australia, is a close relative of the tobacco plant and the authors were able to produce more than 60g of pure griffithsin from a single greenhouse with an area of 5000 square feet. The biophysical characteristics of griffithsin and the nature of the plant host allowed isolation of 99% pure protein after a simple 3-step purification procedure.
The plant-produced protein was found to have broad and potent activity against a panel of primary sexually transmitted HIV-1 isolates representative of viruses prevalent in sub-Saharan Africa, Asia and the West. The recombinant griffithsin was further shown to be non-irritating and non-inflammatory, and to have no mitogenic activity. Since viral entry inhibitors are not commonly used in resource-poor countries, griffithsin produced cost-effectively in Nicotiana benthamiana plants has the potential for prevention and treatment of multi-drug resistant viral infections in developing countries.
Myriad Pharmaceuticals recently announced the acquisition, from Panacos Pharmaceuticals, of bevirimat, a novel HIV-1 maturation inhibitor.
Bevirimat is a derivative of betulinic acid, a triterpenoid isolated from the leaves of the Chinese herb, Syzygium claviflorum, which was found to have potent inhibitory activity against HIV-1. Bevirimat shows potent in vitro activity against a broad range of HIV-1 strains, including isolates that are resistant to drugs currently approved for the treatment of HIV-1: protease inhibitor-resistant HIV-1 strains appear to be especially sensitive to bevirimat. Like HIV-1 protease inhibitors, bevirimat interferes with proteolytic processing of the newly translated viral polyprotein, Gag. One of the last steps in viral maturation is cleavage at the capsid-SP1 junction, and bevirimat is believed to prevent cleavage by binding to the Gag polyprotein at this site. Release of SP1 is essential for proper capsid condensation and function: preventing release of SP1 results in non-infectious virions containing abnormal, unstable cores. Bevirimat-resistant strains of HIV-1 can be generated in vitro, but arise more slowly in strains resistant to protease inhibitors than in wild-type strains. Mutations conferring resistance to bevirimat occur at or near the capsid-SP1 cleavage site.
Clinical studies have shown that bevirimat is well tolerated and have demonstrated significant and clinically relevant reductions in viral load in a subset of patients. Studies have suggested that clinical resistance to bevirimat does not develop rapidly, possibly because of selective pressure to maintain the highly conserved capsid-SP1 cleavage sequence. There may also be a greater hurdle to development of bevirimat resistance in strains of virus that are resistant to protease inhibitors, suggesting that patients with such viruses may be especially likely to benefit from treatment with maturation inhibitors such as bevirimat.
HIV-1 infection is characterized by a wide variety of genetic subtypes in different geographical locations. The predominant subtype found in the western world, clade B, differs substantially from clade C, the main subtype that exists in Africa and Asia. Dementia is common amongst individuals infected with clade B HIV-1, but occurs less frequently amongst individuals infected with clade C HIV-1. Although it had been suspected that the differences in dementia rates were linked to differences in neurotoxicity between the clades, this had been difficult to prove since differences in geographical distribution, differences in host genetics, lifespan following infection or differences in access to antiretrovirals could all affect progression to dementia.
A new study using immunocompromised (SCID) mice has now confirmed the importance of HIV-1 clades in determining the incidence of HIV-associated dementia. The results demonstrate, for the first time, differences in neuropathogenesis between clade B and C HIV-1 isolates in an in vivo model. The study demonstrated that mice infected with representative clade C HIV-1 isolates performed better in cognitive tests than those infected with clade B HIV-1 isolates. Brains of mice exposed to either HIV-1 clade B or HIV-1 clade C showed similar viral loads, but those with clade B HIV-1 showed increased inflammation and neuronal damage.
Using in vitro experiments with purified HIV-1 Tat (Trans-Activator of Transcription) proteins, the team further showed that the differences in neuropathology were linked to structural differences in the Tat protein. Tat from clade C HIV-1 differs from that of other HIV-1 clades in that a highly conserved dicysteine motif (C30-C31) is replaced by a C31S polymorphism.
The authors suggest that targeting HIV-1 Tat may be a way to prevent the neurological effects of AIDS, although this may be difficult to achieve in practice. In the early to mid 1990s, there was considerable interest in developing Tat antagonists but, following the unexpected failure of the selective Tat antagonist Ro 24-7429 to show antiviral activity in clinical studies, interest in the approach appears to have waned.
Telomeres are structures which protect chromosomes during cell division and become progressively shorter as a result; telomerase adds telomere DNA to the ends of chromosomes and increases the potential for cell division. Most non-dividing cells show little or no telomerase activity but telomerase is up-regulated in cells that need to divide repeatedly, such as cells of the immune system. CD8+ T-cells play a crucial role in controlling HIV infection and, in CD8+ T-cells responding to viral antigens, telomerase is up-regulated. During chronic HIV-1 infection, however, CD8+ T-cells lose their ability to up-regulate telomerase, leading to critically short telomeres and reduced antiviral activity. A study jointly carried out by scientists at UCLA and the Geron Corporation has shown that TAT2, a small molecule telomerase activator isolated from the Astragalus root, can prevent or slow telomere shortening and increase antiviral efficacy. CD8+ T-cells from HIV-infected donors treated with TAT2 showed increased proliferation and enhanced ability to fight HIV.
Although telomerase is up-regulated in cancer cells, TAT2 did not enhance telomerase production by tumour cells. Astragalus is also used in traditional Chinese medicine without obvious side effects suggesting that TAT2 will not promote the development of tumours.
The human immunodeficiency virus (HIV) uses host cell surface receptors such as CD4, CCR5 and CXCR4 to gain entry into cells. Recently, monoclonal antibodies and small molecules that block these receptors have joined the armoury of drugs used to combat HIV infection. A new report in the journal Cell describes a genome-wide analysis to search for other virus-host interactions that are important for the early steps of HIV infection.
More than 40 host factors that regulate capsid uncoating and reverse transcription were identified along with 15 cellular factors that facilitate entry of the HIV preintegration complex into the cell nucleus and integration of proviral DNA.
Cytoskeletal proteins and microtubules were found to play roles in early HIV replication, and proteins involved in DNA-damage repair were found to influence the initiation of reverse transcription and the accumulation of HIV-1 DNA products prior to integration. Other cellular systems discovered to play roles in the early stages of HIV infection include nucleic acid binding proteins and the ubiquitin-proteasome pathway. Protein phosphorylation and dephosphorylation events were found to have a possible role in the regulation of HIV-1 reverse transcription, and prostaglandins were shown to have a potential role in HIV-1 nuclear import. Several cellular factors were also found to be important for HIV-1 DNA integration.
The study revealed totally new pathways in HIV infection, including involvement of Notch signalling in reverse transcription. A full understanding of the role of each of these host factors in cell types infected by HIV will be essential to assess their contribution to disease progression. However, given the success of compounds which block viral entry receptors, modulation of the activity of some of the newly discovered pathways could provide novel strategies for the treatment of HIV/AIDS.