ABSTRACT
An efficient combination solution-phase/solid-phase route enabling the diversification of the P1', P2', and P3 subsites of indinavir has been established. The synthetic sequence can facilitate the rapid generation of HIV protease inhibitors possessing more favorable pharmacokinetic properties as well as enhanced potencies. Multiple compound dosing in vivo may also accelerate the identification of potential drug candidates.
Subject(s)
Combinatorial Chemistry Techniques , HIV Protease Inhibitors/chemistry , Indinavir/analogs & derivatives , Indinavir/chemistry , Animals , Cell Line , Dogs , HIV Protease/metabolism , HIV Protease Inhibitors/chemical synthesis , HIV Protease Inhibitors/pharmacokinetics , HIV Protease Inhibitors/pharmacology , Humans , Indinavir/chemical synthesis , Indinavir/pharmacokinetics , Indinavir/pharmacology , Models, Molecular , Molecular Conformation , Molecular Structure , T-LymphocytesABSTRACT
There is compelling evidence that members of the caspase (interleukin-1beta converting enzyme/CED-3) family of cysteine proteases and the cytotoxic lymphocyte-derived serine protease granzyme B play essential roles in mammalian apoptosis. Here we use a novel method employing a positional scanning substrate combinatorial library to rigorously define their individual specificities. The results divide these proteases into three distinct groups and suggest that several have redundant functions. The specificity of caspases 2, 3, and 7 and Caenorhabditis elegans CED-3 (DEXD) suggests that all of these enzymes function to incapacitate essential homeostatic pathways during the effector phase of apoptosis. In contrast, the optimal sequence for caspases 6, 8, and 9 and granzyme B ((I/L/V)EXD) resembles activation sites in effector caspase proenzymes, consistent with a role for these enzymes as upstream components in a proteolytic cascade that amplifies the death signal.
Subject(s)
Apoptosis/physiology , Caspases , Cysteine Endopeptidases/metabolism , Serine Endopeptidases/metabolism , Amino Acid Sequence , Animals , Caenorhabditis elegans/enzymology , Caenorhabditis elegans Proteins , Granzymes , Humans , Mammals , Recombinant Proteins/metabolism , Substrate SpecificityABSTRACT
BACKGROUND: Interleukin-1beta converting enzyme (ICE/caspase-1) is the protease responsible for interleukin-1beta (IL-1beta) production in monocytes. It was the first member of a new cysteine protease family to be identified. Members of this family have functions in both inflammation and apoptosis. RESULTS: A novel method for identifying protease specificity, employing a positional-scanning substrate library, was used to determine the amino-acid preferences of ICE. Using this method, the complete specificity of a protease can be mapped in the time required to perform one assay. The results indicate that the optimal tetrapeptide recognition sequence for ICE is WEHD, not YVAD, as previously believed, and this led to the synthesis of an unusually potent aldehyde inhibitor, Ac-WEHD-CHO (Ki = 56 pM). The structural basis for this potent inhibition was determined by X-ray crystallography. CONCLUSIONS: The results presented in this study establish a positional-scanning library as a powerful tool for rapidly and accurately assessing protease specificity. The preferred sequence for ICE (WEHD) differs significantly from that found in human pro-interleukin-1beta (YVHD), which suggests that this protease may have additional endogenous substrates, consistent with evidence linking it to apoptosis and IL-1alpha production.
