Brian M. McKeever

The three-dimensional structure of human granzyme B compared to caspase-3, key mediators of cell death with cleavage specificity for aspartic acid in P1

BACKGROUND Granzyme B, one of the most abundant granzymes in cytotoxic T-lymphocyte (CTL) granules, and members of the caspase (cysteine aspartyl proteinases) family have a unique cleavage specificity for aspartic acid in P1 and play critical roles in the biochemical events that culminate in cell death. RESULTS We have determined the three-dimensional structure of the complex of the human granzyme B with a potent tetrapeptide aldehyde inhibitor. The Asp-specific S1 subsite of human granzyme B is significantly larger and less charged than the corresponding Asp-specific site in the apoptosis-promoting caspases, and also larger than the corresponding subsite in rat granzyme B. CONCLUSIONS The above differences account for the variation in substrate specificity among granzyme B, other serine proteases and the caspases, and enable the design of specific inhibitors that can probe the physiological functions of these proteins and the disease states with which they are associated.

The differential interactions of peroxisome proliferator-activated receptor gamma ligands with Tyr473 is a physical basis for their unique biological activities.

Despite their proven antidiabetic efficacy, widespread use of peroxisome proliferator-activated receptor (PPAR)γ agonists has been limited by adverse cardiovascular effects. To overcome this shortcoming, selective PPARγ modulators (SPPARγMs) have been identified that have antidiabetic efficacy comparable with full agonists with improved tolerability in preclinical species. The results of structural studies support the proposition that SPPARγMs interact with PPARγ differently from full agonists, thereby providing a physical basis for their novel activities. Herein, we describe a novel PPARγ ligand, SPPARγM2. This compound was a partial agonist in a cell-based transcriptional activity assay, with diminished adipogenic activity and an attenuated gene signature in cultured human adipocytes. X-ray cocrystallography studies demonstrated that, unlike rosiglitazone, SPPARγM2 did not interact with the Tyr473 residue located within helix 12 of the ligand binding domain (LBD). Instead, SPPARγM2 was found to bind to and activate human PPARγ in which the Tyr473 residue had been mutated to alanine (hPPARγY473A), with potencies similar to those observed with the wild-type receptor (hPPARγWT). In additional studies, we found that the intrinsic binding and functional potencies of structurally distinct SPPARγMs were not diminished by the Y473A mutation, whereas those of various thiazolidinedione (TZD) and non-TZD PPARγ full agonists were reduced in a correlative manner. These results directly demonstrate the important role of Tyr473 in mediating the interaction of full agonists but not SPPARγMs with the PPARγ LBD, thereby providing a precise molecular determinant for their differing pharmacologies.

Both Coactivator LXXLL Motif-dependent and -independent Interactions Are Required for Peroxisome Proliferator-activated Receptor γ (PPARγ) Function

Nuclear receptor activation is dependent on recruitment of coactivators, including CREB-binding protein (CBP/p300) and steroid receptor coactivator-1 (SRC-1). A three-dimensional NMR approach was used to probe the coactivator binding interface in the peroxisome proliferator-activated receptor γ (PPARγ) ligand binding domain (LBD). In the presence of a CBP peptide, peaks corresponding to 20 residues in helices 3, 4, 5, and 12 of the LBD were attenuated. Alanine mutants revealed that K301A, V315A, Y320A, L468A, and E471A were required for binding of both CBP and SRC-1 and for cell-based transcription. Several additional amino acids in helix 4 of the PPARγLBD were defective with respect to CBP recruitment, but retained relatively normal SRC-1 recruitment. Thus these amino acid residues may be important determinants of specificity for nuclear receptor LBD interactions with discrete coactivator molecules.

Protein crystal growth results for shuttle flights STS-26 and STS-29

Abstract Recent advances in protein crystallography have significantly shortened the time and labor required to determine the three-dimensional structures of macromolecules once good crystals are available. Crystal growth has become a major bottleneck in further development of protein crystallography. Proteins and other biological macromolecules are notoriously difficult to crystallize. Even when usable crystals are obtained, the crystals of essentially all proteins and other biological macromolecules are poorly ordered, and diffract to resolutions considerably lower than that available for most crystals of simple organic and inorganic compounds. One promising area of research which is receiving widespread attention is protein crystal growth in the microgravity environment of space. A series of protein crystal growth experiments were performed on US shuttle flight STS-26 in September 1988 and STS-29 in March 1989. These proteins had been studied extensively in crystal growth experiments on earth prior to the microgravity experiments. For those proteins which produced crystals of adequate size, three-dimensional intensity data sets with electronic area detector systems were collected. Comparisons of the microgravity-grown crystals with the best earth-grown crystals obtained in numerous experiments demostrate that the microgravity-grown crystals of these proteins are larger, display more uniform morphologies, and yield diffraction data to significantly higher resolutions. Analyses of the three-dimensional data sets by relative-Wilson plots indicate that the space-grown crystals are more highly ordered at the molecular level than their earth-grown counterparts.

Discovery of VTP-27999, an Alkyl Amine Renin Inhibitor with Potential for Clinical Utility.

Structure guided optimization of a series of nonpeptidic alkyl amine renin inhibitors allowed the rational incorporation of additional polar functionality. Replacement of the cyclohexylmethyl group occupying the S1 pocket with a (R)-(tetrahydropyran-3-yl)methyl group and utilization of a different attachment point led to the identification of clinical candidate 9. This compound demonstrated excellent selectivity over related and unrelated off-targets, >15% oral bioavailability in three species, oral efficacy in a double transgenic rat model of hypertension, and good exposure in humans.

Benzimidazolones: A New Class of Selective Peroxisome Proliferator-Activated Receptor γ (PPARγ) Modulators

A series of benzimidazolone carboxylic acids and oxazolidinediones were designed and synthesized in search of selective PPARγ modulators (SPPARγMs) as potential therapeutic agents for the treatment of type II diabetes mellitus (T2DM) with improved safety profiles relative to rosiglitazone and pioglitazone, the currently marketed PPARγ full agonist drugs. Structure-activity relationships of these potent and highly selective SPPARγMs were studied with a focus on their unique profiles as partial agonists or modulators. A variety of methods, such as X-ray crystallographic analysis, PPARγ transactivation coactivator profiling, gene expression profiling, and mutagenesis studies, were employed to reveal the differential interactions of these new analogues with PPARγ receptor in comparison to full agonists. In rodent models of T2DM, benzimidazolone analogues such as (5R)-5-(3-{[3-(5-methoxybenzisoxazol-3-yl)benzimidazol-1-yl]methyl}phenyl)-5-methyloxazolidinedione (51) demonstrated efficacy equivalent to that of rosiglitazone. Side effects, such as fluid retention and heart weight gain associated with PPARγ full agonists, were diminished with 51 in comparison to rosiglitazone based on studies in two independent animal models.

A Role for the Aspartyl Protease from the Human Immunodeficiency Virus Type 1 (HIV-1) in the Orchestration of Virus Assembly

Functional HIV-1 protease (PR) is required for the maturation of viral proteins, for the appearance of characteristic structural features in the virion (as determined by electron microscopy), and for the final assembly of mature virus. Most importantly, HIV-1 PR activity is required for the development of infectivity. Still largely undefined, however, is the timing and control of protease action in this assembly process. Based on the three-dimensional structure of HIV-1 PR2,3 and experimental data reported in the literature, we propose a comprehensive virus assembly model that highlights the role of HIV-1 PR, suggests further experiments to verify the validity of the model, and poses specific questions relevant to the ultimate exploitation of HIV-1 protease as a therapeutic target.

Structure-based design and synthesis of 1,3-oxazinan-2-one inhibitors of 11β-hydroxysteroid dehydrogenase type 1.

Structure based design led directly to 1,3-oxazinan-2-one 9a with an IC(50) of 42 nM against 11β-HSD1 in vitro. Optimization of 9a for improved in vitro enzymatic and cellular potency afforded 25f with IC(50) values of 0.8 nM for the enzyme and 2.5 nM in adipocytes. In addition, 25f has 94% oral bioavailability in rat and >1000× selectivity over 11β-HSD2. In mice, 25f was distributed to the target tissues, liver, and adipose, and in cynomolgus monkeys a 10 mg/kg oral dose reduced cortisol production by 85% following a cortisone challenge.

Spirocyclic ureas: Orally bioavailable 11β-HSD1 inhibitors identified by computer-aided drug design

Structure-guided drug design led to the identification of a class of spirocyclic ureas which potently inhibit human 11beta-HSD1 in vitro. Lead compound 10j was shown to be orally bioavailable in three species, distributed into adipose tissue in the mouse, and its (R) isomer 10j2 was efficacious in a primate pharmacodynamic model.

Optimization of orally bioavailable alkyl amine renin inhibitors.

Structure-guided drug design led to new alkylamine renin inhibitors with improved in vitro and in vivo potency. Lead compound 21a, has an IC(50) of 0.83nM for the inhibition of human renin in plasma (PRA). Oral administration of 21a at 10mg/kg resulted in >20h reduction of blood pressure in a double transgenic rat model of hypertension.