Characterisation of inhibitor binding to the LEDGF/p75-binding domain of HIV-1 integrase
Date
2018
Authors
Harrison, Angela Theresa
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Abstract
Human immunodeficiency virus type 1 (HIV-1) integrase (IN) catalyses the irreversible integration of the viral genome into the host genome with the aid of the host co-factor lens epithelium derived growth factor (LEDGF/p75). LEDGF/p75 protects HIV-1 IN from proteasomal degradation, facilitates transport and directs DNA binding. Allosteric IN inhibitors (ALLINIs) were discovered in 2010 by Christ et al and then several research groups followed suite. ALLINIs were found to cause aberrant multimerisation of HIV-1 IN and have a profound effect on virion maturation. In previous studies lovastatin and cefdinir were identified to be inhibitors of the HIV-1 IN-LEDGF/p75 interaction. In this study, the aim was to characterise the interaction of both compounds in comparison with a known ALLINI, CX05168 with HIV-1 IN, and determine their modes of action. After the expression and purification of the recombinant proteins, the compounds were screened through an AlphaScreen assay to assess their ability to inhibit the HIV-1 IN-LEDGF/p75 interaction. All three of the compounds: lovastatin, cefdinir and CX05168 inhibited the interaction with IC50 values of 1.97 ± 0.28 µM, 4.05 ± 0.18 µM and 1.33 ± 0.5 5µM for subtype B HIV-1 IN and 1.98 ± 0.28 µM, 5.22 ± 0.92 µM and 1.79 ± 0.63 µM for subtype C HIV-1 IN, respectively. The AlphaScreen was then adjusted for the addition of donor DNA. This caused a reduction in the potency of CX05168 and cefdinir but had no effect on lovastatin. The IC50 values for cefdinir and CX05168 increased to 52.02 ± 1.82 µM and 20.46 ± 0.76 µM for subtype B HIV-1 IN and 41.87 ± 0.95 µM and 21.28 ± 0.52 µM for subtype C HIV-1 IN, respectively. This was the first indication that lovastatin had a different mode of action. With the findings that ALLINIs cause HIV-1 IN to aberrantly multimerise follow up tests included the compounds in two separate assays to determine if they caused multimerisation. The first assay was a cross-linking study and the second was differential light scattering assay. The only compound shown to have the ability to cause aberrant multimerisation was CX05168. Follow up tests were conducted to determine if the compounds had any stabilising effect on the HIV-1 IN enzyme. In agreement with the multimerisation assay only CX05168 showed any effect on the stability of HIV-1 IN. In this assay a difference in the potency of CX05168 on the subtype C HIV-1 IN was seen, where less stabilisation occurs. Hydrogen deuterium exchange mass spectroscopy was used to determine binding sites or allosteric changes to HIV-1 IN. Overall visual allosterism on HIV-1 IN subtype B and C was seen with CX05168. Lovastatin had clear binding areas that differed from CX05168, whereas cefdinir showed few areas of binding or similarities. This indicated that all three compounds displayed different binding models and in combination with the other assays confirmed their different modes of action. Surface plasmon resonance was used to determine the binding kinetics. The noise to signal ratio for lovastatin and cefdinir proved to be too high to obtain
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usable results indicating a possible poor binding ability. CX05168 showed good binding in agreement with our previous results but once again a difference in binding the subtype B HIV-1 IN and subtype C HIV-1 IN could be seen. We finalised our tests with cellular screening. Lovastatin proved to be too toxic to the mammalian cells to test antiviral efficiency. Cefdinir and CX05168 showed viral inhibition with CX05168 being less potent against the HIV-1 subtype C as opposed to the HIV-1 subtype B viruses. In conclusion, this study found that the three compounds (lovastatin, cefdinir and CX05168) inhibit the HIV-1 IN-LEDGF/p75 interaction in different ways. Results highlight that CX05168 functions as a LEDGF/p75 IN inhibitor by causing aberrant IN multimerisation, however, it is less potent towards HIV-1 subtype C, which is the predominant subtype circulating in South Africa. Lovastatin and cefdinir inhibit the direct interaction of HIV-1 IN-LEDGF/p75 without multimerisation which decreases the antiviral potency. The compounds of most value are those causing aberrant multimerisation of HIV-1 IN as they have potent downstream effects, lovastatin and cefdinir in their current chemical structure are not promising ALLINI’s, and require structural modifications to fulfil their promise.
Description
A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand, in fulfilment of the requirements for the degree Of Doctor of Philosophy, 2018
Keywords
Lens Epithelium-Derived Growth Factor (LEDGF/p75)