Indole acids as a novel PDE2 inhibitor chemotype that demonstrate pro-cognitive activity in multiple species.

An internal HTS effort identified a novel PDE2 inhibitor series that was subsequently optimized for improved PDE2 activity and off-target selectivity. The optimized lead, compound 4, improved cognitive performance in a rodent novel object recognition task as well as a non-human primate object retrieval task. In addition, co-crystallization studies of close analog of 4 in the PDE2 active site revealed unique binding interactions influencing the high PDE isoform selectivity.

Structural definition and substrate specificity of the S28 protease family: the crystal structure of human prolylcarboxypeptidase.

BACKGROUND: The unique S28 family of proteases is comprised of the carboxypeptidase PRCP and the aminopeptidase DPP7. The structural basis of the different substrate specificities of the two enzymes is not understood nor has the structure of the S28 fold been described. RESULTS: The experimentally phased 2.8 A crystal structure is presented for human PRCP. PRCP contains an alpha/beta hydrolase domain harboring the catalytic Asp-His-Ser triad and a novel helical structural domain that caps the active site. Structural comparisons with prolylendopeptidase and DPP4 identify the S1 proline binding site of PRCP. A structure-based alignment with the previously undescribed structure of DPP7 illuminates the mechanism of orthogonal substrate specificity of PRCP and DPP7. PRCP has an extended active-site cleft that can accommodate proline substrates with multiple N-terminal residues. In contrast, the substrate binding groove of DPP7 is occluded by a short amino-acid insertion unique to DPP7 that creates a truncated active site selective for dipeptidyl proteolysis of N-terminal substrates. CONCLUSION: The results define the structure of the S28 family of proteases, provide the structural basis of PRCP and DPP7 substrate specificity and enable the rational design of selective PRCP modulators.

Expression, purification and crystallization of human prolylcarboxypeptidase.

Prolylcarboxypeptidase (PrCP) is a lysosomal serine carboxypeptidase that cleaves a variety of C-terminal amino acids adjacent to proline and has been implicated in diseases such as hypertension and obesity. Here, the robust production, purification and crystallization of glycosylated human PrCP from stably transformed CHO cells is described. Purified PrCP yielded crystals belonging to space group R32, with unit-cell parameters a = b = 181.14, c = 240.13 A, that diffracted to better than 2.8 A resolution.

Structural basis for the inhibition of RNase H activity of HIV-1 reverse transcriptase by RNase H active site-directed inhibitors.

HIV/AIDS continues to be a menace to public health. Several drugs currently on the market have successfully improved the ability to manage the viral burden in infected patients. However, new drugs are needed to combat the rapid emergence of mutated forms of the virus that are resistant to existing therapies. Currently, approved drugs target three of the four major enzyme activities encoded by the virus that are critical to the HIV life cycle. Although a number of inhibitors of HIV RNase H activity have been reported, few inhibit by directly engaging the RNase H active site. Here, we describe structures of naphthyridinone-containing inhibitors bound to the RNase H active site. This class of compounds binds to the active site via two metal ions that are coordinated by catalytic site residues, D443, E478, D498, and D549. The directionality of the naphthyridinone pharmacophore is restricted by the ordering of D549 and H539 in the RNase H domain. In addition, one of the naphthyridinone-based compounds was found to bind at a second site close to the polymerase active site and non-nucleoside/nucleotide inhibitor sites in a metal-independent manner. Further characterization, using fluorescence-based thermal denaturation and a crystal structure of the isolated RNase H domain reveals that this compound can also bind the RNase H site and retains the metal-dependent binding mode of this class of molecules. These structures provide a means for structurally guided design of novel RNase H inhibitors.