A framework to understand the needs of the medical students of the future.

Purpose: Medical education is undergoing curricular reform driven by internal needs and external pressures. Concurrently, medical students are changing. More diverse student bodies, particularly those from underrepresented minorities, bring different skill sets, needs and priorities to their medical school education. Here we present their voices.Methods: In order to explore the stories, needs and motivations of future physicians, we conducted a small-scale study of students from diverse backgrounds in US medical schools. Our interviews revealed two core themes about medical school: (1) that medical school is an extreme physical and emotional challenge for all students, and (2) that medical school education consists of multiple conflicting goals. Combining the two themes provides a descriptive topology of student's motivations and strategies for getting through medical school. Using this framework, we deconstruct the experience of medical school to reveal the gaps and misalignments between the traditional system of medical education and what medical students want, expect and need to be successful.Conclusions: We discuss the implications of our research and this framework for curricula and the learning environment with a particular focus on (1) cooperative learning and social interdependence theory and (2) an expanded understanding of diversity and the needs of under-represented students.

Endogenous effectors of human liver glycogen phosphorylase modulate effects of indole-site inhibitors.

Phosphorylase is regulated by a number of small-molecular-weight effectors that bind to three sites on the enzyme. Recently, a fourth site referred to as the indole-inhibitor site has been identified. Synthetic compounds bind to the site and inhibit activity. However, the effects of these compounds in the presence of other endogenous effectors are unknown. We have determined the effects of four indole derivative glycogen phosphorylase inhibitors (GPI) on recombinant human liver glycogen phosphorylase a activity. The GPIs tested were all potent inhibitors. However, the endogenous inhibitors (glucose, ADP, ATP, fructose 1-phosphate, glucose 6-phosphate, UDP-glucose) and the activator (AMP) markedly reduced the inhibitory effect of GPIs. Consistent with these in vitro findings, the IC50 for the inhibition of glycogenolysis in cells and the liver drug concentration associated with glucose-lowering activity in diabetic ob/ob mice in vivo were also significantly higher than those determined in in vitro enzyme assays. The inhibitory effect of indole-site effectors is modulated by endogenous small-molecular-weight effectors of phosphorylase a activity. However, at higher concentrations (10-30 microM), the GPI effect was dominant and resulted in inhibition of phosphorylase a activity irrespective of the presence or absence of the other modulators of the enzyme.

5-Chloroindoloyl glycine amide inhibitors of glycogen phosphorylase: synthesis, in vitro, in vivo, and X-ray crystallographic characterization.

The synthesis, in vitro, and in vivo biological characterization of a series of achiral 5-chloroindoloyl glycine amide inhibitors of human liver glycogen phosphorylase A are described. Improved potency over previously reported compounds in cellular and in vivo assays was observed. The allosteric binding site of these compounds was shown by X-ray crystallography to be the same as that reported previously for 5-chloroindoloyl norstatine amides.

Sulfotransferase structural biology and inhibitor discovery.

Sulfotransferases catalyze the transfer of a sulfuryl group from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to proteins, carbohydrates and small molecules. The sulfotransferases comprise cytosolic and Golgi-resident enzymes; Golgi-resident enzymes represent fertile territory for identifying pharmaceutical targets. Structure-based sequence alignments indicate that the structural fold, and the PAPS-binding site, is conserved between the two classes. Initial efforts to identify sulfotransferase inhibitors by screening kinase inhibitor libraries yielded competitive inhibitors of PAPS with muM IC(50) values. Within particular classes of Golgi-resident sulfotransferases that show tight in vitro specificity, the substrate-binding site might be a suitable drug target, although sulfotransferases are generally assumed to be difficult to inhibit as a result of the expected size and chemical character of the substrate-binding site.

Strategies for drug discovery by targeting sulfation pathways.

Posttranslational modifications of proteins such as phosphorylation have been recognized as pivotal modulators of biological activity in healthy and diseased tissues. Sulfation is a key posttranslational modification the role of which in physiology and pathology is only now becoming appreciated. Whereas phosphorylation is central to intracellular signal transduction, sulfation modulates cell-cell and cell-matrix communication. Sulfation involves a class of enzymes known as sulfotransferases, which transfer sulfate from the ATP-like sulfate donor 3'phosphoadenosine-5'phosphosulate to glycoproteins, glycolipids or metabolites. This review focuses on Golgi-localized sulfotransferases, their molecular biology and biochemistry, and strategies towards discovery of sulfotransferase inhibitors that could have potential as therapeutics in inflammation, cancer and infectious diseases.

X-ray crystallographic and kinetic studies of human sorbitol dehydrogenase.

Sorbitol dehydrogenase (hSDH) and aldose reductase form the polyol pathway that interconverts glucose and fructose. Redox changes from overproduction of the coenzyme NADH by SDH may play a role in diabetes-induced dysfunction in sensitive tissues, making SDH a therapeutic target for diabetic complications. We have purified and determined the crystal structures of human SDH alone, SDH with NAD(+), and SDH with NADH and an inhibitor that is competitive with fructose. hSDH is a tetramer of identical, catalytically active subunits. In the apo and NAD(+) complex, the catalytic zinc is coordinated by His69, Cys44, Glu70, and a water molecule. The inhibitor coordinates the zinc through an oxygen and a nitrogen atom with the concomitant dissociation of Glu70. The inhibitor forms hydrophobic interactions to NADH and likely sterically occludes substrate binding. The structure of the inhibitor complex provides a framework for developing more potent inhibitors of hSDH.

Specificity determinants of human cathepsin s revealed by crystal structures of complexes.

Cathepsin S, a lysosomal cysteine protease of the papain superfamily, has been implicated in the preparation of MHC class II alphabeta-heterodimers for antigen presentation to CD4+ T lymphocytes and is considered a potential target for autoimmune-disease therapy. Selective inhibition of this enzyme may be therapeutically useful for attenuating the hyperimmune responses in a number of disorders. We determined the three-dimensional crystal structures of human cathepsin S in complex with potent covalent inhibitors, the aldehyde inhibitor 4-morpholinecarbonyl-Phe-(S-benzyl)Cys-Psi(CH=O), and the vinyl sulfone irreversible inhibitor 4-morpholinecarbonyl-Leu-Hph-Psi(CH=CH-SO(2)-phenyl) at resolutions of 1.8 and 2.0 A, respectively. In the structure of the cathepsin S-aldehyde complex, the tetrahedral thiohemiacetal adduct favors the S-configuration, in which the oxygen atom interacts with the imidazole group of the active site His164 rather than with the oxyanion hole. The present structures provide a detailed map of noncovalent intermolecular interactions established in the substrate-binding subsites S3 to S1' of cathepsin S. In the S2 pocket, which is the binding affinity hot spot of cathepsin S, the Phe211 side chain can assume two stable conformations that accommodate either the P2-Leu or a bulkier P2-Phe side chain. This structural plasticity of the S2 pocket in cathepsin S explains the selective inhibition of cathepsin S over cathepsin K afforded by inhibitors with the P2-Phe side chain. Comparison with the structures of cathepsins K, V, and L allows delineation of local intermolecular contacts that are unique to cathepsin S.

Characterization and mutagenesis of Gal/GlcNAc-6-O-sulfotransferases.

The installation of sulfate groups on the carbohydrate residues of glycoproteins, glycolipids, and glycosaminoglycans is a critical posttranslational modification that occurs in all higher eukaryotes. The Gal/GalNAc/GlcNAc-6-O-sulfotransferases (GSTs) are a recently discovered family of carbohydrate sulfotransferases that share significant sequence homology at the amino acid level and mediate a number of different biological processes such as leukocyte adhesion at sites of chronic inflammation. Structural and mechanistic studies of this family of sulfotransferases have been hindered by the lack of a productive recombinant protein expression system. We developed a baculovirus expression system for five of the seven cloned GSTs and determined their kinetic parameters using both thin-layer chromatography and a recently developed polymer dot-blot assay. We used these tools to perform the first site-directed mutagenesis study of a member of this sulfotransferase family, GST2. Using sequence alignments with other carbohydrate and cytosolic sulfotransferases, we selected residues within the putative binding regions for 3'-phosphoadenosine 5'-phosphosulfate (PAPS) and the carbohydrate substrate for mutagenesis. Kinetic analysis of the mutants identified residues that are essential for catalytic activity. These results should facilitate mechanistic studies and the development of small molecule inhibitors of this enzyme family to ameliorate chronic inflammatory diseases.

Structure-activity analysis of the purine binding site of human liver glycogen phosphorylase.

Human liver glycogen phosphorylase (HLGP) catalyzes the breakdown of glycogen to maintain serum glucose levels and is a therapeutic target for diabetes. HLGP is regulated by multiple interacting allosteric sites, each of which is a potential drug binding site. We used surface plasmon resonance (SPR) to screen for compounds that bind to the purine allosteric inhibitor site. We determined the affinities of a series of compounds and solved the crystal structures of three representative ligands with K(D) values from 17-550 microM. The crystal structures reveal that the affinities are partly determined by ligand-specific water-mediated hydrogen bonds and side chain movements. These effects could not be predicted; both crystallographic and SPR studies were required to understand the important features of binding and together provide a basis for the design of new allosteric inhibitors targeting this site.

Integrated effects of multiple modulators on human liver glycogen phosphorylase a.

Hepatic glucose production is increased in people with type 2 diabetes. Glucose released from storage in liver glycogen by phosphorylase accounts for approximately 50% of the glucose produced after an overnight fast. Therefore, understanding how glycogenolysis in the liver is regulated is of great importance. Toward this goal, we have determined the kinetic characteristics of recombinant human liver glycogen phosphorylase a (HLGPa) (active form) and compared them with those of the purified rat enzyme (RLGPa). The Michaelis-Menten constant (K(m)) of HLGPa for P(i), 5 mM, was about fivefold greater than the K(m) of RLGPa. Two P(i) (substrate) concentrations were used (1 and 5 mM) to cover the physiological range for P(i). Other effectors were added at estimated intracellular concentrations. When added individually, AMP stimulated, whereas ADP, ATP and glucose inhibited, activity. These results were similar to those of the RLGPa. However, glucose inhibition was about twofold more potent with the human enzyme. UDP-glucose, glucose 6-phosphate, and fructose 1-phosphate were only minor inhibitors of both enzymes. We reported previously that when all known effectors were present in combination at physiological concentrations, the net effect was no change in RLGPa activity. However, the same combination reduced HLGPa activity, and the inhibition was glucose dependent. We conclude that a combination of the known effectors of phosphorylase a activity, when present at estimated intracellular concentrations, is inhibitory. Of these effectors, only glucose changes greatly in vivo. Thus it may be the major regulator of HLGPa activity.

Structure and function of the eukaryotic ribosome: the next frontier.

As the catalytic and regulatory centers of protein synthesis in cells, ribosomes are central to many aspects of cell and structural biology. Recent work highlights the unique properties and complexity of eukaryotic ribosomes and their component rRNAs and proteins.