Design of an effective vaccine against HIV must take into account the high degree of variability in the sequence of the envelope proteins that has been observed in clinical isolates, and the high mutation rate of the virus. Vaccines based on the envelope glycoprotein gp120 have been ineffective in protecting against clinical isolates of HIV. Most of the antibodies that are elicited are directed against variable portions of the sequence. Some HIV-infected patients, however, develop neutralizing antibodies with broader strain specificity, many of which appear to be directed at the three- dimensional shape of the CD4 binding site on gp120. This part of the molecule is conserved in structure, because all HIV use the CD4 receptor to gain entry into the cell. Our hypothesis is that this part of gp120 will make a more effective vaccine antigen than the whole protein. We are therefore constructing recombinant or synthetic molecules that display this conformation-dependent structure. We have identified peptide structures that can mimic the sites recognized by some of the neutralizing antibodies using phage-display technology and are displaying these peptides on the surface of other viruses or nanoparticles, and using them to immunize mice and rabbits. We are also taking the synthetic peptide sequences and determining their three-dimensional structures when bound to the antibodies. The information obtained from X-ray crystallography of the peptide-antibody complexes will be used to design better antigens that can elicit a more effective immune response to the virus. Comparing the structures of different antibodies to the CD4 binding site that vary significantly in their ability to neutralize primary HIV strains will also shed light on what determines their potency.
Blocking The Interaction Between the HIV Envelope Protein and the Chemokine Receptors CCR5 and CXCR4:
This project aims to determine the structure of the CCR5 and CXCR4 chemokine receptors at their ligand binding sites and their interaction with agonist and antagonist molecules. CCR5 is the co-receptor required for infection by primary isolates of HIV (the M-tropic/R5 strains predominantly transmitted between people) and CXCR4 is used by the X4 strains that predominate at later stages. The goals of the project are to: (1) Synthesize photo-activatable analogs of peptide and non-peptide antagonists that block HIV infection. (2) Photo-crosslink bioactive analogs to CCR5 or CXCR4, and locate crosslinking sites using mass spectrometry (LC/MS/MS) to map the antagonist binding pocket in the receptor. (3) Map the structure of the HIV/chemokine binding pocket by scanning photoactivatable amino acid analogs over chemokine peptide agonist sequences, photocrosslinking the bioactive analogs to the receptors, and using MS to determine residues lining the binding sites. The ultimate goal is to provide a foundation for more effective structure-based design of HIV entry inhibitors.
Design of small-molecule CXCR4 antagonists for HIV and cancer treatment
We are designing and synthesizing non-peptide small molecule inhibitors of CXCR4. Lead compounds that compete with known CXCR4 antagonists, such as the peptide T-140, are being tested for their ability to inhibit HIV infection in vitro, and to inhibit tumor cell metastasis in vitro and in vivo.