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Cell Kinase Blocks HIV Infection In Vitro; May Have Rx Application

Investigators at the National Institutes of Health and Boston University reported this week that they have prevented HIV infection in vitro by inactivating interleukin-2-inducible T-cell kinase, or ITK.
The Tec family tyrosine kinase regulates T-cell receptor-induced activation of PLCγ-1, Ca2+ mobilization and transcription factor activation, and actin rearrangement downstream of both T-cell receptors and chemokine receptors.  
When the investigators inhibited ITK, they found that they inhibited the HIV life cycle at three different steps: the level of entry, the level of expression, and the level of the virus being released, said Andrew Henderson, an associate professor of medicine and microbiology at Boston University School of Medicine.
The researchers, which used the Jurkat leukemic T-cell line and primary human CD4+ T cells in their work, found that “at each step, we sort of disrupt that process a little bit, and the sum of the whole is greater than the individual parts, so to speak,” said Henderson, who is a co-author on the study.
“ITK seems to be involved in three different steps [of the HIV viral life cycle], which means that it would be very difficult for the virus to mutate and escape the effects of this molecule, because it would have to change three different functions,” he told CBA News this week.   
In addition, if viral proteins are the drug target, they can mutate and the virus can develop resistance, said Henderson. Cellular proteins such as ITK are not as susceptible to mutations, so resistance would not be an issue if cellular proteins are drug targets.
“With that said, this is still a very basic and very preliminary observation,” Henderson said.
Because infecting T-cells with HIV requires T-cell activation, chemokine receptors, and actin reorganization, the researchers looked at whether ITK affects HIV infection using ITK-specific siRNA, a kinase-inactive ITK mutant, and an ITK inhibitor (BMS509744) that Bristol-Myers Squibb previously showed can inhibit ITK.  
In Henderson’s study, online this week in the Proceedings of the National Academy of Sciences, the investigators found that loss of ITK function resulted in a reduction in intracellular p24 levels in T-cells that had been infected with HIV. They also found that after the establishment of HIV infection, loss of ITK function decreased the spread of the virus within the culture. 

“ITK seems to be involved in three different steps [of the HIV viral life cycle], which means that it would be very difficult for the virus to mutate and escape the effects of this molecule.”

Inhibition of ITK partially blocked HIV viral entry into T cells, an effect that correlated with decreased actin polarization to gp120, the scientists said. However, expression of the HIV CXCR4 and CD4 coreceptors was not affected by ITK inhibition.
In addition, the researchers found that ITK was required for efficient HIV transcription, and overexpression of ITK increased viral transcription and virus-like particle formation.
Members of Pamela Schwartzberg’s lab at the National Human Genome Research Institute were the co-investigators on the study. In terms of future studies, Schwartzberg’s lab is “very much interested in ITK and its role in immunity, and the role it plays in diseases like asthma,” said Henderson. “That is their major focus.”
Henderson went on to say that his lab in particular is interested in HIV, and “we are focused on elucidating how ITK is facilitating the viral life cycle at these three different steps. We are focusing on the entry block and the exiting block, and trying to better understand ITK’s role in that process.” 
“We are trying to understand how the chemistry and molecular biology of ITK influences the virus,” Henderson explained.
If Henderson and his colleagues can understand how ITK facilitates the viral life cycle — even if ITK does not turn out to be a very good target — maybe the proteins that it regulates would be good targets, he said.
To determine if ITK is a viable, druggable target “would take people who are interested in drug development and drug design,” said Henderson, adding that he hopes this research will get others excited, and they “will look at, possibly, a compound library and say, ‘OK, let’s test this in the context of HIV.’” 
Henderson said that he does not see ITK inhibition replacing highly active antiretroviral therapy. “I think that it would complement or be a part of HAART. That is one way to look at it — rather than targeting the viral proteins you would be targeting the cellular activity.”
Another company that is looking for ways to identify novel mechanisms of action for antiretroviral drugs is German gene-synthesis firm Geneart (see CBA News, 4/18/08). The company recently won a patent from the German Patent and Trademark Office for its antiviral drug-screening system based on synthesized, virus-like reporter genes. 
Marcus Graf, operations manager and head of Geneart’s gene-synthesis business, and his colleagues reengineered the gene from the jellyfish Aequorea victoria that encodes the green fluorescent protein so that it transfers its mRNA from the nucleus to the cytoplasm of a cell in a manner similar to that of the HIV virus.
Small molecules that interfere with this mRNA export machinery will result in cells that do not light up in an optical detection system or a fluorescence-activated cell sorter.
It is likely that if a drug candidate can block this pathway and prevent the synthetic HIV-like gene from transferring its material to the cytoplasm, “you can be sure that you would also have interfered with the viral replication machinery,” Geneart CEO and CSO Ralf Wagner told CBA News last month.
Like antiretorvirals that inhibit ITK, “anitretrovirals targeting the RNA export machinery could become a complementary therapy to those targeting viral enzymes,” Wagner said.

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