Hans J. Schramm

HIV-1 reproduction is inhibited by peptides derived from the N- and C-termini of HIV-1 protease

Two octapeptides derived from the sequence of the N- and C-termini of HIV-1 protease were tested for their ability to inhibit HIV-1 reproduction. Weak inhibitory activity was found with each of the two peptides. It is assumed that HIV-1 protease is the target of the inhibitory action. In spite of the moderate inhibitory activity the results are encouraging since they may be improved by various means.

Lipopeptides as dimerization inhibitors of HIV-1 protease

Abstract In AIDS therapy, attempts have been made to inhibit the virus-encoded enzymes, e.g. HIV-1 protease, using active site-directed inhibitors. This approach is questionable, however, due to virus mutations and the high toxicity of the drugs. An alternative method to inhibit the dimeric HIV protease is the targeting of the interface region of the protease subunits in order to prevent subunit dimerization and enzyme activity. This approach should be less prone to inactivation by mutation. A list of improved ‘dimerization inhibitors’ of HIV-1 protease is presented. The main structural features are a short ‘interface’ peptide segment, including non-natural amino acids, and an aliphatic N-terminal blocking group. The high inhibitory power of some of the lipopeptides [e.g. palmitoyl-Tyr-Glu-Leu-OH, palmitoyl-Tyr-Glu-(L-thyronine)-OH, palmitoyl-Tyr-Glu-(L-biphenyl-alanine)-OH] with low nanomolar Ki valuesin the enzyme test suggests that mimetics with good bio-availability can be derived for AIDS therapy.

Design of dimerization inhibitors of HIV-1 aspartic proteinase: A computer-based combinatorial approach

Inhibition of dimerization to the active form of the HIV-1 aspartic proteinase (HIV-1 PR) may be a way to decrease the probability of escape mutations for this viral protein. The Multiple Copy Simultaneous Search (MCSS) methodology was used to generate functionality maps for the dimerization interface of HIV-1 PR. The positions of the MCSS minima of 19 organic fragments, once postprocessed to take into account solvation effects, are in good agreement with experimental data on peptides that bind to the interface. The MCSS minima combined with an approach for computational combinatorial ligand design yielded a set of modified HIV-1 PR C-terminal peptides that are similar to known nanomolar inhibitors of HIV-1 PR dimerization. A number of N-substituted 2,5-diketopiperazines are predicted to be potential dimerization inhibitors of HIV-1 PR.

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.

Cleavage of phosphorylase kinase and calcium-free calmodulin by HIV-1 protease

Phosphorylase kinase and calcium-free calmodulin are digested by human immunodeficiency virus-1 protease. In phosphorylase kinase, the alpha subunit is preferentially hydrolyzed at arg748-val749. The beta subunit is cleaved only slowly at leu678-pro679, and calmodulin, the integral delta subunit of phosphorylase kinase, is not cleaved at all. However, free calmodulin in the calcium-depleted form showed to be a good substrate for the protease. Here the cleavage occurs at phe65-pro66 and met71-met72. This fast hydrolysis of free calmodulin can be blocked by micromolar concentrations of Ca2+ or millimolar concentrations of Mg2+.

COMPUTER AVERAGING OF SINGLE MOLECULES OF TWO FORMS OF α2-MACROGLOBULIN AND THE α2-MACROGLOBULIN/TRYPSIN COMPLEX

Electron micrographs of active cq-macroglobulin (u,M) usually show either almost spherical structures with a diameter of approximately 20 nm and without distinct features or a form similar to the Russian letter ‘‘W with a size of 17 x 10 nm, while the trypsin complex is described as a more compact structure (for a list of references see Ref. 1). In order to improve the significance of electron micrographs of q M , the technique of computer averaging of single particles was applied to profiles found in images of a nearly inactive F-form.’ Furthermore, a preliminary averaging of molecules of the a,M/trypsin complex was performed. i n our electron micrographs of the investigated F-form (FIG. la) most of the molecules show one of the two forms: a compact, nearly round structure with “spokes” and high contrast, and a long, narrow structure with little contrast; the two structural forms are called here the “closed” and the “open” forms. Sets of molecular images were computer averaged; the averaged forms are shown in FIGURES 2a and b. The structures prove that drastic rearrangements can occur in q M molecules. So far, the following three structural forms of %M are the best documented: (a) the unstructured form: the active form of %M most likely has no well-defined shape’; (b) the closed form seems to be the structure of the F-form, probably a more denatured structure with cleaved chains and without activity, which shows the most cage-like shape of the known forms; (c) the open form is probably identical to the described “M”-form often attributed to the active form. It is not clear, however, whether it is still active. Electron micrographs (FIG. lb) of an a,M/trypsin complex preparation show several distinctly different forms usually of low symmetry. Among them are some molecules with a clear structural similarity to the open type, but with four “knobs” in a row. The result of an averaging of 19 visually selected molecules of this type is shown in FIGURE 2c. The additional knobs can be interpreted as the bound trypsin molecules. They lie very close to each other (confirming the results of Ref. 3) and demonstrate the binding of the proteinases in the central part of cqM. Furthermore, the structure obtained of the trypsin complex is not derived from the more cage-like closed form, but from the open form. Even if this averaged form does not represent the main form of the complex, it is certainly one of the forms occurring. Its relatively open structure indicates that purely noncovalent entrapment of proteinases by q M may not be sufficient for reliable binding. Therefore, the main mode of attachment

Inhibition of HIV-1 Replication by Peptidic Protease Inhibitors

During viral replication, the HIV–protease is responsible for post-translational processing of Gag and Gag-Pol polyprotein precursors [14]. The inhibition of this enzyme leads to the generation of immature non-infectious viruses and thereby to a reduction of viral spread [13]. Thus, protease inhibitors are currently the most promising drugs for the HIV therapy, especially in combination with inhibitors of the reverse transcriptase [8,10,13]. However, the protease inhibitors used so far, e. g. in the highly active anti-retroviral therapy (HAART), showed a limited capability to penetrate the cellular plasma membrane. Moreover, they exhibit a high affinity for plasma proteins resulting in a reduced in vivo efficacy, a high dose requirement [7] and toxicity. Most of these pseudo-peptides are active site inhibitors and selective for the HIV-1 protease. However, the application in therapy frequently leads to a rapid development of viral resistance [10]. These disadvantages may be overcome by inhibitors targeting alternative sites of the HIV-i protease.