User:Memechoi/sandbox/Zinc in the treatment of HIV/AIDS

Zinc in the treatment of HIV/AIDS refers to both the direct intake of zinc and use of zinc fingers in gene therapy for the management and prevention of human immunodeficiency virus (HIV) infection and acquired immunodeficiency syndrome (AIDS). It is a current topic of research due to the necessity of zinc in maintaining human health.

HIV/AIDS is a disease typically characterized by a long incubation period and a compromised immune system in the human host, which renders them more vulnerable to other diseases, including opportunistic infections and cancer. AIDS specifically refers to a consequence of HIV infection, resulting in lowered CD4+ T-cell counts beyond a certain threshold or the presence of diseases associated with the condition. Two main strains are present in humans: HIV-1 and HIV-2, although the former is more infectious and common; as such, most research is focused on neutralizing HIV-1.

Presently, there are no known cures for HIV/AIDS and the main method of treatment involves use of anti-retroviral drug therapy, which is expensive and laden with side effects. Nevertheless, near-restoration of immune system response is possible, although immunological discordance (unexpectedly high or no change in T-cell levels) may also occur in 15-30% of patients. Research into HIV-1 vaccines is also underway, although the high rate of mutation and protection of the viral envelope make development of effective antibodies difficult. However, a 2014 clinical in Thailand showed a 30% reduction in infection.

Alternative medicine involving a variety of supplements such as selenium and vitamins are also available for patients although their efficacy has not been proven. Studies by the World Health Organization (WHO) found that supplementation of vitamin A, zinc and iron can produce unfavourable effects in HIV positive adults.

Zinc and its role in biology
See also: Zinc

Zinc (Zn) is only common in its +2 oxidative state, where it typically coordinates tetrahedrally. It is important in maintaining basic cellular functions such as DNA replication, RNA transcription, cell division and cell activation, all of which are necessary for immunologic mechanisms. However, having too much or too little zinc can cause these functions to be compromised.

As biosystems are unable to store zinc, regular intake is necessary. Excessively low zinc intake can lead to zinc deficiency, which can negatively impact an individual's health. This is due to the weakening of the immune system, as zinc is a major component in many enzymes that regulate the immune system.

Zinc deficiency affects about 2.2 billion people around the world and is associated with an increased susceptibility to infectious disease such as HIV/AIDS. The recommended daily intake of zinc for adults is approximately 15-40 milligrams, while for children the value is around 10 milligrams. Zinc deficiency in children can cause growth retardation, delayed sexual maturation, increased infection susceptibility, and diarrhea. However, excess consumption above 80 mg of zinc can cause other disorders such as ataxia, lethargy, and copper deficiency.

Due to the importance of zinc for immunity and its many benefits in various infectious disease, there is more research being done in regards to the relation between zinc and HIV/AIDS.

Mechanisms of action
The main methods by which zinc may be used in treating HIV are by oral zinc supplementation or through gene therapy with zinc finger endonucleases. The effectiveness of supplementation is inconclusive overall, although several mechanisms have been postulated from in vitro studies and the observation that zinc deficiency is seen in HIV positive individuals. In general, zinc may affect HIV via (1) direct activity on HIV and its enzymes, (2) effects on the immune system, or a combination of both factors. The use of zinc finger nucleases focuses on preventing infection of the T-cells that are targeted by the virus. As with zinc supplementation, the treatment is still being evaluated for use in humans.

Effects on HIV
Due to zinc's ubiquity in biosystems, use of supplements may affect HIV positively or negatively. Zinc acts as a cofactor for several important proteins required for viral propagation. Many of these involve a zinc finger motif, such as HIV nucleocapsid proteins, which are chaperones for folding stable viral RNA complexes and require the cation to function. Another example is HIV integrase, which had activity that was 5–15 times faster with the ion despite not requiring zinc to function. Overall, it is uncertain if the blood concentration of zinc may positively affect the activity of viral enzymes and its replication cycle while in vivo.

Another experiment conducted on human HIV-infected cell cultures found the use of zinc to inhibit HIV growth, although the actual mechanism is unknown. Viral propagation was slowed by lowering production of capsid proteins and by lowering HIV RNA transcription. Severe reductions in p24 capsid proteins and viral RNA was found to occur with 100 μg/mL of ZnCl2 with no ill effects to the cells until 1000 μg/mL. A notable point was that only viral RNA transcription was negatively affected by zinc, while the cell was able to operate regularly. It was postulated that the HIV tat transcription protein is affected by zinc, although this hypothesis was contradicted by another paper that found that incubation with zinc did not affect tat’s activity.

Other studies focus on particular enzymes required for HIV replication and the possible mechanisms by which they may be inhibited by zinc.

HIV-1 protease
One such enzyme that was found to be inhibited by zinc is the homodimeric HIV-1 protease, which is used in gene expression. Polyproteins, which are polypeptides containing the amino acid sequence of multiple products, are cleaved by the enzyme to produce complete proteins that can then fold into their functional conformation; without this process, the viral enzymes would be inactive and the infection’s progress would be halted. The enzyme is classed as an aspartic protease due to the presence of a pair of closely associated aspartate residues at the active site. The asparates are necessary in the first step of catalysis where they act as a general base to activate a nucleophile (usually water) which can then attack the peptide bond.

It has been found that addition of zinc into a buffer system containing the enzyme lowered its activity linearly with increasing concentration. The mechanism was a combination of competitive and non-competitive inhibition, depending on the type of substrate used, with non-competitive inhibition corresponding to an actual peptide sequence found in HIV polyproteins. The inhibition was reversible, as addition of the chelating agent EDTA restored the protease’s activity to its normal limits. In addition, the ability of zinc to act as an inhibitor depended on solution pH, with higher effectiveness seen at pH greater than 7 (basic). Other aspartic proteases, such as rennin, pepsin and HIV-2 protease were tested and found to have been inhibited by zinc as well, leading to the conclusion that the enzymes were affected in part due to binding to the active site.

Although the mechanism is not known, one study based on the previous experiment conducted with computer modelling techniques suggested that zinc is four-coordinate to both aspartates via the oxygens. The increasing inhibition at higher pH is rationalized as both residues being completely deprotonated, leaving a larger amount of charge concentrated at the oxygens, which more readily binds to the positively charged zinc. In addition, no exchange of aspartate oxygen ligands occurs, which suggests the configuration is stably bound together. The rest of the protein is unaffected by the zinc bound at the active site, with the network of hydrogen bonds connecting the protease together remaining intact. Another study suggests that the protease’s active site accepts binding of tetrahedrally coordinated metal ions since it resembles the tetrahedral transition state that is present when peptide bonds are cleaved.

HIV reverse transcriptase
Reverse transcriptases (RT) are a class of enzymes that produce a complementary DNA strand from an RNA template. In retroviruses, RT transcribes the single-stranded RNA genome into single-stranded DNA, which acts as a template for construction of a double-stranded segment of DNA; the DNA can then be integrated into the host cell chromosomes. HIV-RT possesses both polymerase and ribonuclease-H capabilities, both of which require divalent cations (metal ions with a +2 charge) as an essential cofactor in the mechanism of catalysis. At physiological conditions, magnesium  acts as the cofactor, but other divalent cations, such as nickel and copper, are available for nucleotide catalysis. However, some divalent cations can also inhibit magnesium-dependent RT activity, including zinc and manganese.

Zinc is an effective inhibitor of many viral polymerases, amongst other enzymes. There are different mechanisms in which zinc can act as an inhibitor, which includes competing for binding sites, allosteric inhibition, or interactions with other enzyme-associated molecules.

A 2011 experiment using enzyme kinetics showed that RT possessed both polymerase and ribonuclease-H activity when zinc was used as the cofactor. Although the enzyme would function at lower concentrations of zinc compared to magnesium, the rates of catalysis were approximately 35 times slower. In addition, only a small amount of zinc was needed to reduce enzyme activity, relative to when larger concentrations of magnesium were present. The authors concluded that zinc was an effective inhibitor of reverse transcriptase when magnesium is present due to the severe decrease in catalysis rates. Although the complete mechanism is unknown, it is possible that zinc's displacement of magnesium at the active site leads to a stable complex with decreased catalytic activity. The authors also note that the concentrations used in the experiment (μM) could lead to adverse health effects in human systems.

Effects on the immune system
As HIV-infected individuals are commonly suffering from zinc deficiency, supplementation may also bolster the immune system by providing the body with enough of the metal to maintain production and activity of enzymes dependent on zinc. One example is thymulin, a zinc-dependent enzyme which has a role in facilitating T-cell maturation. In order to maintain its active conformation, the nonapeptide requires coordination to zinc through two serines through their side-chain and a terminal asparagine via its carboxylate group, with the last position occupied by a water molecule. In addition, zinc also has roles in activating immune system responses such as recruitment of leukocytes to infected areas and phagocytosis of pathogens. The general improvement of the individual's health may increase resistance to opportunistic infections and decrease other negative symptoms associated with zinc deficiency.

Zinc finger nuclease
Main article: Zinc finger nuclease

The zinc finger nuclease is a specific endonuclease designed to bind and cleave at specific positions in the target DNA. There are two domains in which the zinc finger nuclease uses for the binding and cleaving mechanism. The first domain is the DNA binding domain, which consists of eukaryotic transcription factors and contain the zinc finger. The second domain is the nuclease domain, which consists of the FokI restriction enzyme and is responsible for the catalytic cleavage of DNA. For the treatment of HIV, zinc finger nucleases participate in disruption of the CCR5 gene and is delivered using a recombinant adenoviral vector. The process of DNA repair by nonhomologous end joining is prone to error and results in a non-functional gene.

CCR5 genes code for chemokine receptors that are normally found in CD4+ T-cells and participate in the defense against pathogens. However, in HIV infection the virus can bind to the receptor and allow entry into the T-cells. Some individuals have a mutated CCR5 gene that make them resistant to HIV.

The zinc finger motif located in the DNA binding domain allows the nuclease to bind to a specific base sequence approximately three base pairs long. The motif consists of tandem repeats of two cysteines and two histidine fingers bound to a zinc (II) atom arranged in a tetrahedral conformation. In recent studies they have added more fingers to specify larger and rarer cleavage targets.

The FokI catalytic domain must dimerize in order to cleave the DNA at the targeted site, and requires there to be two adjacent zinc finger nucleases (see picture), which independently bind to a specific codon at the correct orientation and spacing. As a result, the two binding events from the two zinc finger nuclease enables specific DNA targeting.

Specificity of genome editing is important in order for the zinc finger nuclease to be a successful application. The consequence of off-targeting cleavage can lead to a decrease in efficiency of the on-target modification in addition to other unwanted changes.

Studies
Zinc supplementation studies showed varied results with some finding a positive effect on HIV-infected patients while others found no effect. The positive effects were improved T-cell counts and a reduction in immunological deterioration. Overall, the effectiveness of supplementation is inconclusive and in need of further studies. The general conclusion from the zinc finger nuclease studies showed that the zinc finger nuclease was effective in vitro. Current research is undergoing to determine if zinc finger modified cells are safe and effective for use in humans.

Zinc supplementation
Zinc deficiency occurs in more then 50% of patients affected by HIV. In clinical studies by Baum et al. consisting of randomized and controlled groups of 231 adults affected with HIV, plasma levels of zinc were found to be lower than 0.75 mg/L. For 18 months, women and men received 12 mg and 15 mg of zinc supplements daily, respectively. It was found that the rate of immunological failure was reduced by 4 times compared to the placebo (control) group. In addition, there was a reduction in the incidence of diarrhea for the group that used the supplements. However, zinc supplementation did not show any results for patients that were on the antiretroviral treatment.

Another random study by Mochegiani et al. was conducted on 57 patients at the late stages of HIV infection. The experiment was performed on adults (who had low CD4+ levels between 250-400 x 10-6 cells/L, and low zinc plasma concentrations of 76-80 ug/L) that were diagnosed with HIV for at least 3 months. They were followed for two years. Among these infected people they were assigned either no supplementation or 45.5 mg of zinc per day. The group that did not take the supplements demonstrated continuing decreases in CD4+ levels, zinc plasma concentration and body weight, whereas the adults that were treated showed an increase in all these factors. Also, the supplementation group showed an increase in the levels of active thymulin. Overall, supplementation appeared to increase factors associated with healthy patients, and decrease the likelihood of other immune infections. Overall, supplementation appeared to increase factors associated with healthy patients, and decrease the likelihood of other immune infections. Nevertheless, no controls with placebo or uninfected groups were used.

Zinc-finger nuclease
In a study performed by ''Perez et. al'', zinc-finger nucleases (ZFN) were designed to disrupt the CCR5 gene, which is a receptor for HIV, in CD4+ cells. The benefits associated with ZFN CCR5 disruption were obtained through experimentation in mice. The mice were infected with HIV and then observed for 50 days. The results of the experiment demonstrated an increase in CD4+ T cells and lower plasma viremia for the group with the ZFN CD4+ cells compared to the untreated group. These results indicated that this method creates resistance to HIV as the ZFN-treated cells block the initial HIV infection and replicate the mutation into their daughter cells.

In the study performed by ''Holt et. al'', zinc finger nucleases were used to disrupt the CCR5 gene in human HSC (hematopoietic stem/progenitor cells). Mice were then used to determine the beneficial role of this disruption. Untreated and ZFN human HSC were transplanted into mice, who were then infected with HIV-1 BAL after 8-12 weeks. The CD4:CD8 ratio was then determined for both groups of mice, with the untreated mice experiencing loss of CD4+ cells, while the ZFN group retaining pre-infection levels. Other observations demonstrated loss of human lymphocytes in the intestines of the untreated group and no difference in the ZFN group. The results of this study along with the data of an AIDS patient who after undergoing HSC transplantation (The Berlin Patient) was cured of his infection, demonstrates the potential long-term control of HIV using this method.

In the study performed by ''Badia et. al'', a zinc finger nuclease was designed that targeted the CCR5 gene and mimics the naturally occurring 32 bp deletion mutation commonly found in humans. This ZNF was found to affect the reading frame of the CCR5 gene, which prevented protein production. These cells were also found to be resistant to infection by HIV (BaL strain). Further analysis also revealed ZFN not having any off-target activity, which demonstrates high specificity of this zinc finger nuclease resulting in its potential to be used in human trials.