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Purpose and Methods Trop-2 is a glycoprotein over-expressed in many solid tumors but at low levels in normal human tissue, providing a potential therapeutic target. We conducted a phase 1 dose-finding study of PF-06664178, an antibody-drug conjugate that targets Trop-2 for the selective delivery of the cytotoxic payload Aur0101. The primary objective was to determine the maximum tolerated dose and recommended phase 2 dose. Secondary objectives included further characterization of the safety profile, pharmacokinetics and antitumor activity. Eligible patients were enrolled and received multiple escalating doses of PF-06664178 in an open-label and unblinded manner based on a modified continual reassessment method. Results Thirty-one patients with advanced or metastatic solid tumors were treated with escalating doses of PF-06664178 given intravenously every 21 days. Doses explored ranged from 0.15 mg/kg to 4.8 mg/kg. Seven patients experienced at least one dose limiting toxicity (DLT), either neutropenia or rash. Doses of 3.60 mg/kg, 4.2 mg/kg and 4.8 mg/kg were considered intolerable due to DLTs in skin rash, mucosa and neutropenia. Best overall response was stable disease in 11 patients (37.9%). None of the patients had a partial or complete response. Systemic exposure of PF-06664178 increased in a dose-related manner. Serum concentrations of free Aur0101 were substantially lower than those of PF-06664178 and total antibody. No correlation of Trop-2 expression and objective response was observed, although Trop-2 overexpression was not required for study entry. The intermediate dose of 2.4 mg/kg appeared to be the highest tolerated dose, but this was not fully explored as the study was terminated early due to excess toxicity. Conclusion PF-06664178 showed toxicity at high dose levels with modest antitumor activity. Neutropenia, skin rash and mucosal inflammation were dose limiting toxicities. Findings from this study may potentially aid in future antibody drug conjugate design and trials.

In a search for more effective anti-diabetic treatment, we used a process coupling low-affinity biochemical screening with high-throughput co-crystallography in the design of a series of compounds that selectively modulate the activities of all three peroxisome proliferator-activated receptors (PPARs), PPARalpha, PPARgamma, and PPARdelta. Transcriptional transactivation assays were used to select compounds from this chemical series with a bias toward partial agonism toward PPARgamma, to circumvent the clinically observed side effects of full PPARgamma agonists. Co-crystallographic characterization of the lead molecule, indeglitazar, in complex with each of the 3 PPARs revealed the structural basis for its PPAR pan-activity and its partial agonistic response toward PPARgamma. Compared with full PPARgamma-agonists, indeglitazar is less potent in promoting adipocyte differentiation and only partially effective in stimulating adiponectin gene expression. Evaluation of the compound in vivo confirmed the reduced adiponectin response in animal models of obesity and diabetes while revealing strong beneficial effects on glucose, triglycerides, cholesterol, body weight, and other metabolic parameters. Indeglitazar has now progressed to Phase II clinical evaluations for Type 2 diabetes mellitus (T2DM).

Protein kinases are a large family of cell signaling mediators undergoing intensive research to identify inhibitors or modulators useful for medicine. As one strategy, small-molecule compounds that bind the active site with high affinity can be used to inhibit the enzyme activity. X-ray crystallography is a powerful method to reveal the structures of the kinase active sites, and thus aid in the design of high-affinity, selective inhibitors. However, a limitation still exists in the ability to produce purified kinases in amounts sufficient for crystallography. Furthermore, kinases exist in different conformation states as part of their normal regulation, and the ability to prepare crystals of kinases in these various states also remains a limitation. In this study, the c-Abl, c-Src, and c-Met kinases are produced in high yields in Escherichia coli by using a bicistronic vector encoding the PTP1B tyrosine phosphatase. A 100-fold lower dose of the inhibitor, Imatinib, was observed to inhibit the unphosphorylated form of c-Abl kinase prepared by using this vector, compared to the phosphorylated form produced without PTP1B, consistent with the known selectivity of this inhibitor for the unactivated conformation of the enzyme. Unphosphorylated c-Met kinase produced with this vector was used to obtain the crystal structure, at 2.15-A resolution, of the autoinhibited form of the kinase domain, revealing an intricate network of interactions involving c-Met residues documented previously to cause dysregulation when mutated in several cancers.

Steroidogenic factor-1 (SF-1) and liver receptor homologue-1 (LRH-1) belong to the fushi tarazu factor 1 subfamily of nuclear receptors. SF-1 is an essential factor for sex determination during development and regulates adrenal and gonadal steroidogenesis in the adult, whereas LRH-1 is a critical factor for development of endodermal tissues and regulates cholesterol and bile acid homeostasis. Regulatory ligands are unknown for SF-1 and LRH-1. A reported mouse LRH-1 structure revealed an empty pocket in a region commonly occupied by ligands in the structures of other nuclear receptors, and pocket-filling mutations did not alter the constitutive activity observed. Here we report the crystal structures of the putative ligand-binding domains of human SF-1 at 2.1-A resolution and human LRH-1 at 2.5-A resolution. Both structures bind a coactivator-derived peptide at the canonical activation-function surface, thus adopting the transcriptionally activating conformation. In human LRH-1, coactivator peptide binding also occurs to a second site. We discovered in both structures a phospholipid molecule bound in a pocket of the putative ligand-binding domain. MS analysis of the protein samples used for crystallization indicated that the two proteins associate with a range of phospholipids. Mutations of the pocket-lining residues reduced the transcriptional activities of SF-1 and LRH-1 in mammalian cell transfection assays without affecting their expression levels. These results suggest that human SF-1 and LRH-1 may be ligand-binding receptors, although it remains to be seen if phospholipids or possibly other molecules regulate SF-1 or LRH-1 under physiological conditions.

An automated high-throughput dispenser has been developed for the setup of protein crystallization trials by vapor diffusion or Microbatch methods. The Hydra-Plus-One is composed of a Hydra-PP system equipped with a motorized XYZ-platform, 96 precision glass syringes and a single-channel microsolenoid dispenser, which transfers 100 nl-50 micro l of protein solution with an accuracy of > 90% at a speed of 60s per 96 wells. Up to 300 micro l of premixed cocktails can be aspirated with the 96-syringe-assembly and dispensed into reservoir and droplet wells within 60s. The Hydra-Plus-One combines high precision, reliability and speed in a cost-effective high-throughput system ideally suited for protein crystallization

The crystallization facility of the TB (Tuberculosis) structural genomics consortium, one of nine NIH sponsored p50 structural genomic centres, provides TB consortium members with automated crystallization, data collection and basic molecular replacement (MR) structure solution up to bias minimized electron density maps. Crystallization setup of up to ten proteins per day follows the CRYSTOOL combinatorial screen protocol using a modular and affordable robotic design with an open architecture. Components include screen preparation, plate setup, automated image acquisition and analysis, and optimisation design. A new 96 well crystallization plate has been designed for optimal robotic handling while maintaining ease of manual crystal harvesting. Robotic crystal mounting, screening, and data collection are conducted in-house and at the Advanced Light Source (ALS) in Berkeley. A simple automated protocol based on MR and homology based structure prediction automatically solves modestly difficult problems. Multiple search models are evaluated in parallel MR and the best multi-segment rigid body refined MR solution is subjected to simulated annealing torsion angle molecular dynamics using CNS, bringing even marginal MR solutions within the convergence radius of the subsequent highly effective bias removal and map reconstruction protocol, Shake&wARP, used to generate electron density for initial rebuilding. Real space correlation plots allow rapid assessment of local structure quality. Modular design of robotics and automated scripts using publicly available programs for structure solution allow for efficient high throughput crystallography - at a reasonable cost.

Staphylococcal enterotoxins (SEs) are superantigenic protein toxins responsible for a number of life-threatening diseases. The X-ray structure of a staphylococcal enterotoxin A (SEA) triple-mutant (L48R, D70R, and Y92A) vaccine reveals a cascade of structural rearrangements located in three loop regions essential for binding the alpha subunit of major histocompatibility complex class II (MHC-II) molecules. A comparison of hypothetical model complexes between SEA and the SEA triple mutant with MHC-II HLA-DR1 clearly shows disruption of key ionic and hydrophobic interactions necessary for forming the complex. Extensive dislocation of the disulfide loop in particular interferes with MHC-IIalpha binding. The triple-mutant structure provides new insights into the loss of superantigenicity and toxicity of an engineered superantigen and provides a basis for further design of enterotoxin vaccines.

Cystalysin is a C(beta)-S(gamma) lyase from the oral pathogen Treponema denticola catabolyzing L-cysteine to produce pyruvate, ammonia and H(2)S. With its ability to induce cell lysis, cystalysin represents a new class of pyridoxal 5'-phosphate (PLP)-dependent virulence factors. The crystal structure of cystalysin was solved at 1.9 A resolution and revealed a folding and quaternary arrangement similar to aminotransferases. Based on the active site architecture, a detailed catalytic mechanism is proposed for the catabolism of S-containing amino acid substrates yielding H(2)S and cysteine persulfide. Since no homologies were observed with known haemolysins the cytotoxicity of cystalysin is attributed to this chemical reaction. Analysis of the cystalysin-L-aminoethoxyvinylglycine (AVG) complex revealed a 'dead end' ketimine PLP derivative, resulting in a total loss of enzyme activity. Cystalysin represents an essential factor of adult periodontitis, therefore the structure of the cystalysin-AVG complex may provide the chemical basis for rational drug design.