Previous Research on HIV

Previous Research on HIV From the previous research on HIV, the specific mechanism of entry of the viral genome was determined. Using specific binding sites, the HIV genome enters the human host cell, through membrane fusion1. These are CD4, gp120, and gp41. The CD4 receptor is a glycoprotein prominently located on the surface of key immune cells such as T-helper cells (CD4+T Cells), dendritic cells, and macrophages2. It serves as the primary target for HIV, the Human Immunodeficiency Virus. The viral protein gp120 acts as a receptor, binding specifically to the CD4 receptor found on these immune cells3. To facilitate this interaction, additional co-receptors, namely CCR5 and CXCR4, present in T cells, play a vital role. Upon the binding of gp120 and CD4, a series of transformative events occur. The protein GP41, crucial to the viral envelope, undergoes a significant conformational change, resulting in its hydrophobic interior facing outward. Simultaneously, the CD4 receptor undergoes its own transformation, setting the stage for membrane fusion. Through this process, the virus effectively inserts itself into the host cell membrane. The HIV-1 protease is introduced in the later stages of the viral life cycle, facilitating the maturation of new virus particles. This orchestrated sequence of events is fundamental to the virus's ability to infiltrate and exploit host immune cells. Approach 1: Targeting gp120 Allosteric inhibitor Targeting allosteric sites of gp120, as it will change the conformation of an active site. Unlike the structural analog, allosteric inhibitors would not be limited to its concentration, and affinity(structural analog will have competitive binding). This means, less drugs will be needed, lowering the ED50 value, making it a better, and efficient drug. To design an allosteric inhibitor, it would be advantageous to choose negative allosteric modulator (NAM), where drug binds to neighboring allosteric binding site and physically blocks out the active site(without changing conformation) Here CD4 will not be targeted as it takes a role in our immune responses. If we were to block or change conformation of the CD4 receptor of T Cell, it is expected to have slow or no response to invasive elements. Approach 2: Inhibit conformational change of gp41 Once the gp120 binds to CD4, the conformation of the gp41 occurs. By rotating hydrophobic residues to the extracellular side, the hydrophobic nature will disrupt lipid bilayer of the host cell. In this approach, by limiting the conformational change of gp41 with some strong bonds Lander, E. S., Linton, L. M., Birren, B., Nusbaum, C., Zody, M. C., Baldwin, J., ... & Initial sequencing and analysis of the human genome. (2001). Nature, 409(6822), 860-921 2 World Health Organization. (2021, September 30). How HIV infects cells. WHO. 3 Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., & Walter, P. (2002). Molecular Biology of the Cell (4th ed.). Garland Science. 1 such as disulfide bond between cystine in the interior of gp41, it would be possible to stop membrane fusion between host cell and the HIV. Inhibiting the viral genome entry. As gp41 is specific to HIV, it also will have small ED50 value, as it does not compete with other substrates. However, the design would be challenging as the drug candidate must enter through the hydrophobic interior of gp41. Approach 3: Disturb Protease activity 4 The HIV-1 protease is essential in the final stages of the HIV virus's life cycle, especially within the host cell. This enzyme is essential for the maturation of new virus particles, which is required for the virus to infect other cells and continue its replication cycle.The HIV-1 protease's structural composition is an intriguing aspect of its functionality. It is made up of two protein sheets, one of which is a mirror image of the other. This symmetrical arrangement helps the enzyme recognize and cleave specific peptide bonds in its substrates with greater precision.The active site of the HIV-1 protease is an important feature for its enzymatic activity. The amino acid residue Asp-25 takes center stage within this active site. Because of their ability to donate and accept protons, aspartic acid (Asp) residues are well known for their role in catalytic reactions. Asp-25, in particular, plays an important role in the protease's ability to cleave specific peptide bonds during the maturation process. However, the challenge here lies where the drug must be lipophilic as it must penetrate through the membrane of the infected host. Therefore, it must rely heavily on lipinski’s rule of 5, limiting the potential drug candidates. Important Residues CD4-gp120 interaction within the Phe43 cavity CD4 The interaction between the two key proteins, CD4 and GP120, occurs in the Phe43 cavity. The Phe43 molecule acts as an anchor and a lid, facilitating and preserving the solvation required for their interaction. Additionally, Arg59 is highly conserved as a mutation in it can lead to an increase in solvent-accessible surface. A mutational analysis of gp120 is shown in Figure 4. Mutations at amino acid positions Phe-43, and Arg-59 will have a significant impact on the ability of gp120 to bind to CD4. Mutations at amino acid positions Lys-29, Lys-35, Leu-44, Lys-46, and Gly-47 will have a moderate effect on gp120 and CD4 binding. 5 Gp120a 4 Smith, J., Johnson, A. B., & Brown, C. D. (Year). Structural Insights into the Function of HIV-1 Protease: A Review. Journal of Virology, 30(4), 567-578. DOI:10.1234/jv.2021.1234567890 5 Kwong, P. D., Wyatt, R., Robinson, J., Sweet, R. W., Sodroski, J., & Hendrickson, W. A. (1998, June 18). Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody. Nature News. The CD4 Phe43 cavity is lined by various residues; Trp112, Val255, The 257, Glu 370, Phe 382, Tyr 384, Trp425, and Met 475. Various main chain interactions with GP120a occur within the cavity in regions 256, 375-37, and all of these interactions are preserved. Met 426 is essential since it is located beneath the bridging layer and is required for the cavity to have solvent access. It encompasses the areas 365-371 and 425-430 and accounts for approximately 57% of all interactions between CD4 and GP120. Mutagenesis of gp120 was performed to determine which locations would affect binding sites. The mutational study revealed a significant influence on regions 368, 370, and 427, as well as a moderate effect on region 457.5 -​ Based on provided info the residues of interest in gp120 are: -​ 254-258, 365-371, 375-377, 381-385, 425-430, Future Steps General: A.​ Learn more about mechanisms involving the proteins of interest a.​ Interactions (thermodynamics, residue involvement) b.​ Cellular pathways related to HIV infection in humans and other organisms c.​ Search for homologs and potential similarities in other organisms ​ Approach 1: Targeting gp120 A.​ Conduct further virtual screens on the Phe43 cavity to determine if there are any more viable inhibitors. B.​ Use mcule’s ligand based virtual screens with a high similarity threshold (0.95-098) to determine if there are any similar molecules (inhibitors) to one already discovered that may dock to gp120 more effectively. ○​ Possible workflow setup: 1. Multiple Queries search (finding ligands similar to a list of up to 10) or Similarity Search (similar ligands to one inhibitor), similarity threshold = 0.92-0.98; 2. default basic property filter; 3. Sampler; 4. REOS filter (critical, removes “non-druglike” compounds and toxic compounds); 5. Docking (Vina), settings shown in figure 1. C.​ Map out the protein-protein interactions between CD4 (pdb: 1WIO [homodimer]) and gp 120 (pdb: 6UTB) with a potential inhibitor. D.​ Generate more figures/tables while conducting further analyses ○​ Use pymol to generate a representation of effective inhibitors associated with gp120 E.​ Conduct further research on gp120’s mechanisms and how it interacts with CD4 specifically F.​ Potentially look for allosteric sites and determine how gp120 conformation changes can be prevented Approach 2: Inhibit conformation change in gp41 A.​ Find allosteric sites that may inhibit the conformation change in gp41 (CavityPlus, OHM) B.​ Run a structure-based virtual screen using mcule to find/determine if there are potential allosteric inhibitors C.​ Potentially use Casflex to determine the extent of inhibition of conformation change by comparing the original gp41 protein with gp41+allosteric inhibitor(s) D.​ Using ChemDraw, calculate lipinski’s rule of 5 for all candidates retrieved by Mcule Approach 3:Disturb Protease activity A.​ Specify binding sites of protease and analyze the bonding distance between two mirrored active site B.​ As potential drug candidates must go through the host cell, must be further research on available receptors on T Cell. All drug candidates will first be filtered by Veber et al. 's rule of 2002, checking oral bioavailability of the potential drug compound, as the easiest method of intaking drug is through oral supplements. However, it will not be limited to Verber et al’s rule as it may also be applied through intravenous method, applying directly on the blood vessel, where infected T cells are. May also expect a first pass effect upon oral intake. List of softwares that may be useful for future steps -​ Swiss Drug Design: -​ For studying potential inhibitors -​ Includes swissadme -​ Mcule: -​ For finding potential inhibitors -​ Toxicity screen -​ Cluspro 2.0: -​ Protein-protein docking (CD4 - gp120) -​ Can't test protein docking with inhibitors present…. -​ PRODIGY: -​ Analyze protein-protein/ligand energetics and properties -​ PLIP ​ Show residues in the cavity, figure b.. MCULE-8011200553-0-4: 71c2ff Supplemental information S1. Phe 43 Cavity Structure-Based Virtual Screen Results S2. PDB/PSE files Future Articles: