Jutta Hansen

HIV protease inhibitor HOE/BAY 793, structure-activity relationships in a series of C2-symmetric diols.

A detailed structure-activity relationship of C2-symmetric diol inhibitors of HIV-1 protease leads to inhibitor 6 (HOE/BAY 793) which is outstanding in the inhibition of the enzyme and in the inhibition of viral replication in HIV infected cell culture (IC50: 0.3 nM; EC50: 3 nM). There are well defined steric requirements for the design of the side chains P1-P3 of the inhibitors. In addition, all three side chains need to be lipophilic. While the enzyme tolerates hydrophilic substituents in some cases, drastic reductions in anti-HIV activity are observed in cell culture, most likely due to insufficient cell penetration.

Epitope Mapping and Functional Characterization of Monoclonal Antibodies Specific for Herpes Simplex Virus Type I DNA Polymerase

Three monoclonal antibodies (MAbs 1051a, 1051b and 1051c) were raised against a surface region (residues 597-686) of the herpes simplex virus type 1 (HSV-1) DNA polymerase (HSV pol), and their epitopes were mapped. The MAbs reacted serotype specifically with the native and denatured HSV pol, as shown by Western blot analysis, immunoprecipitation and immunofluorescence microscopy, indicating their usefulness for biochemical studies and clinical diagnosis of HSV-1 infections, MAb 1051c, displaying the least cross-reactivity with cellular proteins in the Western blot analysis, was successfully utilized not only for coimmunoprecipitation, but also for the analysis and three-dimensional modeling of the cellular sites of HSV pol interaction by confocal laser immunofluorescence microscopy.

Inhibition of HIV-1 protease by short peptides derived from the terminal segments of the protease

The active HIV-1 protease is a homodimeric enzyme. A beta-sheet consisting of N- and C-terminal segments provides the main driving force for dimerization of the inactive protomers. Several short peptides with sequences derived from the N- and C-termini of the protease were tested for inhibition of protease activity and for inhibition of HIV-1 replication in lymphocytes. Medium inhibitory activity was found with each of the peptides in the enzyme test and no inhibition of the lymphocytes was found up to 200 micrograms/ml. The enzyme tests indicate that HIV-1 protease is the target of the inhibitory action. Synergistic action could not be found with pairs of the peptides derived from the two different termini. Prolonged incubation with one of the peptides increased inhibition indicating a slow dissociation of the protease dimers. No cytotoxic effect of the inhibitors could be found below 200 micrograms/ml.

A Novel Protein Tag from Herpes Simplex Virus Type 1 DNA Polymerase

An epitope (HPOL) derived from the so-called thumb region of the herpes simplex virus type 1 DNA polymerase in combination with a monoclonal antibody (MAb 1051c) was tested for protein tagging. Using a conventional expression vector, a DNA cassette encoding the HPOL epitope was fused to the C-terminus of the dihydrofolate reductase (DHFR) gene such that the recombinant DHFR contained both a N-terminal HIS-tag and a C-terminal HPOL tag. Expression of recombinant DHFR in Escherichia coli cells was compared by Western blot analysis using either mouse RGS·HIS antibody or MAb 1051c. Immunostaining revealed that both antibodies reacted specifically with DHFR, but the detection sensitivity achieved with MAb 1051c was about 15-fold greater using a standard staining protocol. An HPOL antibody column was successfully applied for affinity purification of DHFR, demonstrating the usefulness of the HPOL epitope/MAb 1051c system for protein tagging, expression monitoring and purification of HPOL-tagged recombinant proteins.

Structure-activity relationships in a series of C2-symmetric HIV-protease inhibitors

Publisher Summary This chapter describes a series of inhibitors in which a central building block, containing a transition-state mimetic and the P1 and P1’ residues of the HIV-PR cleavage site, is placed in a C 2 -symmetrical peptidic environment and it presents a detailed structure-activity relationship (SAR) for both enzyme inhibition and in vitro activity. The synthesis of the symmetric diamino diol moiety of HOE/BAY 793 is accomplished either by reductive pinacol coupling of N-protected phenylalaninal with SmI 2 or using a mannitol-based bisepoxide which is reacted with (C 6 H 5 ) 2 CuLi. Starting from this central building block, the side chains containing P2, P2’, P3, P3’ are introduced using standard peptide chemistry. These prodrugs release HOE/BAY 793 in a cyclization reaction, which is either only chemically driven or which can be preceded by an enzymatic cleavage. Regarding the inhibition of HIV-PR the phosphinic acid containing inhibitors behave very similar to the diol containing inhibitors with respect to the filling of the P1, P2, and P3 sites.