STRUCTURE-BASED DESIGN OF LIGANDS FOR PROTEIN BASIC DOMAINS - APPLICATION TO THE HIV-1 TAT PROTEIN

A methodology has been developed for designing ligands to bind a flexi ble basic protein domain where the structure of the domain is essentia lly known. It is based on an empirical binding free energy function de veloped for highly charged complexes and on Monte Carlo simulations in internal coordinates with both the ligand and the receptor being flex ible. HIV-1 encodes a transactivating regulatory protein called Tat. f inding of the basic domain of Tat to TAR RNA is required for efficient transcription of the viral genome. The structure of a biologically ac tive peptide containing the Tat basic RNA-binding domain is available from NMR studies. The goal of the current project is to design a ligan d which will bind to that basic domain and potentially inhibit the TAR -Tat interaction. The basic domain contains six arginine and two lysin e residues. Our strategy was to design a ligand for arginine first and then a superligand for the basic domain by joining arginine ligands w ith a linker. Several possible arginine Ligands were obtained by searc hing the Available Chemicals Directory with DOCK 3.5 software. Phytic acid, which can potentially bind multiple arginines, was chosen as a b uilding block for the superligand. Calorimetric binding studies of sev eral compounds to methylguanidine and Arg-/Lys-containing peptides wer e performed. The data were used to develop an empirical binding free e nergy function for prediction of affinity of the ligands for the Tar b asic domain. Modeling of the conformations of the complexes with both the superligand and the basic domain being flexible has been carried o ut via Biased Probability Monte Carlo (BPMC) simulations in internal c oordinates (ICM 2.6 suite of programs). The simulations used parameter s to ensure correct folding, i.e., consistent with the experimental NM R structure of a 25-residue Tat peptide, from a random starting confor mation. Superligands for the basic domain were designed by joining tog ether two molecules of phytic acid with peptidic and peptidomimetic li nkers. The Linkers were refined by varying the length and side chains of the linking residues, carrying out BPMC simulations, and evaluation of the binding foe energy for the best energy conformation. The disso ciation constant of the best ligand designed is estimated to be in the low- to mid-nanomolar range.