Thomas A. Rano

A Combinatorial Approach Defines Specificities of Members of the Caspase Family and Granzyme B FUNCTIONAL RELATIONSHIPS ESTABLISHED FOR KEY MEDIATORS OF APOPTOSIS

There is compelling evidence that members of the caspase (interleukin-1β 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 andCaenorhabditis 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.

A combinatorial approach for determining protease specificities: application to interleukin-1beta converting enzyme (ICE).

BACKGROUNDnInterleukin-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.nnnRESULTSnA 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.nnnCONCLUSIONSnThe 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.

Solid phase synthesis of aryl ethers via the mitsunobu reaction

Abstract A procedure for the preparation of aryl ethers on a solid support employing the Mitsunobu reaction is described. Either polymer bound phenols or benzyl alcohols react rapidly and cleanly with TMAD/Bu 3 P and the appropriate electrophile/nucleophile to provide the aryl ether in excellent yield and purity after cleavage from the solid support.

A highly efficient method for the preparation of 2-aryl substituted carbapenems exploiting a Pd(0) mediated cross-coupling reaction

Abstract A remarkably mild procedure for the synthesis of 2-aryl substituted carbapenems via a palladium catalyzed coupling reaction of a vinyl triflate with aryl stannanes is described. Employing Pd2(DBA)3.CHCl3 as the catalyst and tris(2,4,6-trimethoxyphenyl)phosphine as the ligand provides generous yields of the desired β-lactams. Reaction times are brief while reaction temperatures never exceed ambient.

Indinavir analogues with blocked metabolism sites as HIV protease inhibitors with improved pharmacological profiles and high potency against PI-resistant viral strains.

Indinavir analogues with blocked metabolism sites show highly improved pharmacokinetic profiles in animals. The cis-aminochromanol substituted analogues exhibited excellent potency against both the wild-type (NL4-3) virus and protease inhibitor-resistant HIV strains.

Combinatorial Diversification of Indinavir: In Vivo Mixture Dosing of an HIV Protease Inhibitor Library

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.

HIV protease inhibitors with picomolar potency against PI-Resistant HIV-1 by extension of the P3 substituent.

A biaryl pyridylfuran P(3) substituent on the hydroxyethylene isostere scaffold affords HIV protease inhibitors (PIs) with picomolar (IC(50)) potency against the protease enzymes from PI-resistant HIV-1 strains. Inclusion of a gem-dimethyl substituent afforded compound 3 with 100% oral bioavailability (dogs) and more than double the t(1/2) of indinavir. Inhibition of multiple P450 isoforms is dependent on the regiochemistry of the pyridyl nitrogen in these compounds.