PRELIMINARY CRYSTALLOGRAPHIC STUDY OF NATIVE HIV-1 PROTEASE INHIBITED BY HYDROXYETHYLAMINE MODIFIED TETRAPEPTIDES OF Boc-Phe-[ ( R / S )-CH ( OH ) CH 2 NH ]-Phe-Ile-Phe-NH 2 TYPE

The HIV-1 protease is essential for replication of infective virus HIV, and therefore is an attractive target for the design of specific inhibitors. In search for new inhibitors, substantial effort is devoted to understanding the nature of the inhibitor binding modes in the active site, using X-ray diffraction on crystals as the primary source of structural information. This paper describes the crystallization and preliminary diffraction study of HIV-1 (BRU) protease complexed with the Boc-Phe[(S)-CH(OH)CH2NH]-Phe-Ile-Phe-NH2 inhibitor (SI). The SI is a four-amino-acid pseudopeptidic inhibitor, where the scissile peptide bond is replaced by the (2-hydroxyethyl)amine (HEA) isostere. Current state of crystallization of HIV-1 protease complexed with RI, (Boc-Phe[(R)-CH(OH)CH2NH]-Phe-Ile-Phe-NH2), which is a stereoisomer of SI, is also reported. First crystallization trials were based on crystallization studies performed with complexes of HIV-1 PR with inhibitors SE, RE, RQ. These inhibitors differ from SI in the amino acid at P2’ position, carrying Glu or Gln instead of Ile, and in configuration at C4, the carbon bearing hydroxygroup of the HEA group. The hanging-drop vapor diffusion technique has been used in all experiments. In the case of Glu/Gln containing inhibitors protein-inhibitor mixture of 3 mg/ml HIV PR, 0.544 mM inhibitor (four-fold molar excess over protease) in 50 mM sodium acetate pH 5.6, 1 mM EDTA, 0.05% 2-sulfonylethan-1-ol ( -mercaptoethanol, 5% DMSO was used. The optimum crystallization conditions found for these crystals are: 1M NH4H2PO4, 100 mM sodium citrate, pH 4.5, temperature 6 8 C [1]. In contrast to Glu/Gln containing inhibitors, SI inhibitor addition caused protein precipitation even in the absence of a salt precipitant. A probable reason is higher hydrophobicity of SI inhibitor. Addition of ammonium phosphate under the conditions described above produced no crystal growth and no additional precipitation. Crystals of maximum dimensions 0.5x0.05x0.05 mm were grown at pH 6-8, 25 C and low precipitant concentration (0.1 M NH4H2PO4, 5 mM sodium citrate, 50 mM sodium acetate), but reproducibility of the crystal growth was very low and crystals often coexisted with precipitate. Good reproducibility has not been achieved until the following measures have been undertaken to decrease protease precipitation by inhibitor: 1) decreasing the inhibitor concentration to a two-fold molar excess over protease 2) increasing the concentration of DMSO in protease-inhibitor mixture to 10% 3) avoiding an abrupt decrease in DMSO concentration in the hanging drop after mixing protein solution with the reservoir liquid 4) including 0.05% TritonX-100 in drop as well as in reservoir solution helped to prevent the crystal twinning that elsewhere became a problem at this stage 5) increasing the protein concentration resulted in a major improvement of reproducibility These measures lead to two different crystal forms (needles and platelets). NaCl was used as precipitant in all but initial experiments.