Antiviral strategy targeting Gag polyprotein

http://www.drexelmed.edu/Home/AboutOurFaculty/SimonCocklin.aspx

HIV-1 is a small virus that expresses only 16 proteins. In order for HIV-1 to replicate and cause disease, its proteins must interact with and usurp the normal functions of host cell proteins. This interplay between viral and host proteins is evident at virtually every step in the HIV replication cycle, from binding and entry to particle release. Research in my laboratory is focused upon two main areas of HIV-1 research: small-molecule inhibitor discovery and the identification and investigation of host cell proteins that critically interact with the HIV-1 Gag protein. We have adopted a multidisciplinary approach to researching these areas, combining data from computational, biochemical, biophysical, virological, and structural investigations to achieve our goals. We actively collaborate with research groups at Drexel University College of Medicine, University of Alabama at Birmingham, University of Pennsylvania, Johns Hopkins University, Dana Farber Cancer Institute (Harvard University) and the Southern Research Institute.

Current Major Projects

Cellular biology and host cell protein interactions of the HIV-1 MA protein: The Gag polyprotein is a structural protein that plays a central role in the late stages of viral replication.

The Gag polyprotein consists of several domains, three of which are functionally conserved among retroviruses:

• the nucleocapsid (NC) domain;
  
• the capsid (CA) domain;
  
• and the myristoylated matrix (MA) domain.
  

The HIV-1 MA protein, encoded as the N-terminal portion of Gag, is a small, multifunctional protein responsible for regulating various stages of the viral replication cycle. Functional studies have revealed that the matrix protein (MA) of HIV-1 is critically involved in key processes including the intracellular localization of the Gag polyprotein, the incorporation of the viral envelope glycoprotein into virus particles, and early postentry events.

Moreover, several lines of research indicate these roles may be dependent upon MA interacting with host proteins, yet relatively little is known about the identity or role of these cellular co-factors. Several studies have either directly or indirectly demonstrated the interaction of MA with a number of cellular factors. These include the minor phospholipid, phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2], AP-2, and AP-3 clathrin adaptor complexes; HO3, a histidyl-tRNA synthetase, the translation elongation factor 1-α; the polycomb group protein embryonic ectoderm development (EED); the suppressor of cytokine signaling 1 (SOCS1) protein; calmodulin (CaM); and the adenosine diphosphate ribosylation factor (Arf) proteins.

Given the breadth of functions of the HIV-1 MA protein in both the early and late stages of the viral life cycle, we believe that MA may participate in a larger number of interactions with host cell factors than previously appreciated and that each interaction may itself modulate the interactions that the HIV-1 MA protein is capable of by alteration of the structure of the protein. We have recently performed an exhaustive yeast two-hybrid (Y2H) screen, using HIV-1 MA as bait, and against a cDNA library from primary human leukocytes and activated mononuclear cells. This screen has resulted in the identification of a number of novel host cell protein interactors, in addition to the discovery of proteins previously demonstrated to be required for HIV-1 replication. Interestingly, a common theme among the hits is the participation in intracellular protein transport processes. We are currently confirming and investigating these hits with a view to an improved understanding of the role of the HIV-1 MA protein within the HIV-1 replication cycle and the possibility of identifying new critical interactions that may be targeted therapeutically.

Small-molecule inhibitor discovery: Combination anti-HIV therapy, commonly referred to as highly active antiretroviral therapy, has led to a dramatic reduction in mortality and morbidity in HIV-infected patients. Currently more than 25 antiretroviral drugs are available to treat HIV infection. The majority of them target the HIV-1 reverse transcriptase (RT) and protease enzymes. Recently, antiretroviral drugs that inhibit the viral integrase, the six-helix bundle core formation of the gp41 transmembrane protein, or the host cell protein CCR5 required for virus–cell fusion have been approved for the clinic. Despite these successes, current therapies for HIV-1 are limited by the development of multidrug-resistant virus and by significant cumulative drug toxicities. The development of new classes of antiretroviral agents with novel modes of action is therefore highly desirable and is a driving force for the pursuit of small-molecule inhibitors of other, more difficult viral targets, such as the viral regulatory and accessory proteins. We are currently exploring small-molecule targeting of the HIV-1 Gag (matrix and capsid domains) as an antiviral strategy.

Figure 1. Field point representation of first generation CA inhibitor compound, I-XW-053, generated using FieldTemplater (Cresset BioMolecular Discovery, Welwyn Garden City, Hertfordshire, UK; www.cresset-group.com). Blue field points (spheres) highlight energy minima for a positively charged probe, red for a negative probe. Yellow spheres represent an attractive van der Waals minima for a neutral probe and orange spheres represent hydrophobic centroids. Oxygen atoms are shown in red, nitrogen in blue. The size of the points is related to the strength of the interaction.

Small-molecule modulation of the HIV-1 capsid (CA) protein―The HIV-1 capsid (CA) is an essential viral protein that performs two major roles in the life cycle of HIV-1: one structural, in which it forms a protein shell that shields both the viral genome and the replicative enzymes of HIV-1, and the other regulatory, in which the precise temporal disassembly of this shell coordinates postentry events such as reverse transcription. The CA protein is composed of two domains: the C-terminal domain (CTD) and the N-terminal domain (NTD). Both of these domains make critical inter- and intradomain interactions that are critical for the formation of the capsid shell. The NTD of the capsid protein is the structural anchor for the formation of the hexameric lattice by which the HIV-1 capsid assembles. The stability of this hexameric lattice, which is also conferred by the NTD, regulates the precise temporal series of replicative events after fusion; capsids that are too stable or too unstable do not enter into reverse transcription correctly. Therefore, in theory, any compound that disrupts the normal interactions of the capsid—whether by inhibiting assembly, accelerating disassembly, or artificially stabilizing the core—should attenuate the virus. The essential roles played by capsid within the HIV-1 life cycle, coupled with the existence of known compounds with the ability to disrupt CA-CA interactions, make the capsid’s hexamerization interface a new, attractive therapeutic target. As such, we have employed a virtual screening strategy to identify small molecules with the potential to alter assembly of HIV-1 CA by perturbation of the N-terminal domain (NTD-NTD) hexamerization interface. This strategy has resulted in the identification of a number of compounds, which after size reduction and optimization of their physical-chemical properties, inhibited the replication of a diverse panel of primary HIV-1 isolates in peripheral blood mononuclear cells (PBMCs), while displaying no appreciable cytotoxicity. This antiviral activity was restricted to HIV-1 as determined by cytopathic effect assays against a panel of DNA- and RNA-based viruses. The direct interaction of the compounds with the HIV-1 CA protein has been quantified using surface plasmon resonance (SPR) and isothermal titration calorimetry. Moreover, SPR studies using CA proteins mutated in the compounds’ proposed binding region confirm that residues involved in the NTD-NTD interface are required for interaction. We are currently applying medicinal chemistry approaches to improve the efficacy of these compounds.

Figure 2. Surface rendering of HIV-1 MA showing the residues that form the collective compound binding site, along with the docked structures of selected compound hits.

Targeting the HIV-1 matrix (MA) protein phosphoinositide [4,5] bisphosphate (PIP2)-binding site―The HIV-1 matrix (MA) protein is a structural protein critically involved in both pre- and postintegration stages in the life cycle of the retrovirus. The HIV-1 MA protein has long been known to be crucial for virion assembly, functioning to target assembly to the plasma membrane and facilitating the incorporation of the envelope glycoproteins, gp120 and gp41, into nascent virions. The details regarding its precise function in the early stages of HIV-1 replication are less well defined.

Binding of MA to phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] and the conformational changes that this interaction elicits are key steps in the replication of HIV-1. The high degree of conservation of this region, coupled with its modulatory effect on the structure of the MA protein, makes the MA PI(4,5)P2-binding site a potentially attractive antiviral target. As such, we initiated a structure-based in silico screen to identify novel small molecules with the potential to bind to HIV-1 MA within this region. This approach yielded the identification of four compounds that bind to the HIV-1 MA protein (as judged by surface plasmon resonance and nuclear magnetic resonance) and inhibit the replication of primary HIV-1 isolates in PBMCs with half-maximal inhibitory concentration (IC50) values of 9 to 30 µM. These compounds display specificity to retroviruses, inhibiting HIV-1 and simian immunodeficiency virus (SIV), while showing no effect on other DNA- and RNA-based viruses and little or no cytotoxicity up to 100 µM. Moreover, these four compounds can be grouped into three classes: early-stage inhibitors, late-stage inhibitors, and one compound that appears to disrupt both early and late events. With these exciting results now in hand, we are further investigating their precise mode of action and applying medicinal chemistry approaches to improve the efficacy of these compounds.