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The N-terminal fragment 1-34 of parathyroid hormone (PTH), administered intermittently, results in increased bone formation in patients with osteoporosis. PTH and a related molecule, parathyroid hormone-related peptide (PTHrP), act on cells via a common PTH/PTHrP receptor. To define more precisely the ligand-receptor interactions, we have crystallized human PTH (hPTH)-(1-34) and determined the structure to 0.9-A resolution. hPTH-(1-34) crystallizes as a slightly bent, long helical dimer. Analysis reveals that the extended helical conformation of hPTH-(1-34) is the likely bioactive conformation. We have developed molecular models for the interaction of hPTH-(1-34) and hPTHrP-(1-34) with the PTH/PTHrP receptor. A receptor binding pocket for the N terminus of hPTH-(1-34) and a hydrophobic interface with the receptor for the C terminus of hPTH-(1-34) are proposed.

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The crystal structures of four active site-directed thrombin inhibitors, 1-4, in a complex with human alpha-thrombin have been determined and refined at up to 2.0 A resolution using X-ray crystallography. These compounds belong to a structurally novel family of inhibitors based on a 2,3-disubstituted benzo[b]thiophene structure. Compared to traditional active-site directed inhibitors, the X-ray crystal structures of these complexes reveal a novel binding mode. Unexpectedly, the lipophilic benzo[b]thiophene nucleus of the inhibitor appears to bind in the S1 specificity pocket. At the same time, the basic amine of the C-3 side chain of the inhibitor interacts with the mostly hydrophobic proximal, S2, and distal, S3, binding sites. The second, basic amine side chain at C-2 was found to point away from the active site, occupying a location between the S1 and S1' sites. Together, the aromatic rings of the C-2 and C-3 side chains sandwich the indole ring of Trp60D contained in the thrombin S2 insertion loop defined by the sequence "Tyr-Pro-Pro-Trp." [The thrombin residue numbering used in this study is equivalent to that reported for chymotrypsinogen (Hartley BS, Shotton DM, 1971, The enzymes, vol. 3. New York: Academic Press. pp 323-373).] In contrast to the binding mode of more classical thrombin inhibitors (D-Phe-Pro-Arg-H, NAPAP, Argatroban), this novel class of benzo[b]thiophene derivatives does not engage in hydrogen bond formation with Gly216 of the thrombin active site. A detailed analysis of the three-dimensional structures not only provides a clearer understanding of the interaction of these agents with thrombin, but forms a foundation for rational structure-based drug design. The use of the data from this study has led to the design of derivatives that are up to 2,900-fold more potent than the screening hit 1.

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Human non-pancreatic secretory phospholipase A(2) (hnps-PLA(2)) is a group IIA enzyme that is massively over-expressed in a variety of severe inflammatory diseases. The enzyme degrades membrane phospholipids and it has been hypothesized that this activity can lead to a loss of tissue and organ integrity and function. This report overviews efforts directed toward the identification and clinical evaluation of a new class of anti-inflammatory drugs that specifically targets and inhibits the catalytic site of this hydrolytic enzyme. To achieve this goal, structure-based drug design was applied to a lead molecule identified by random high volume screening. Through an iterative process consisting of X-ray structure determination followed by inhibitor modification and testing, the lead compound was improved more than 6000-fold. Detailed information learned from earlier X-ray studies of stable substrate mimics aided this inhibitor improvement process. The optimized drug candidate, LY315920/S-5920, is currently undergoing phase II clinical evaluation. The outcome of studies such as these will define with greater clarity the pathological role of hnps-PLA(2) in human inflammatory diseases.

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Mutations in the obese gene (OB) or in the gene encoding the OB receptor(OB-R) result in obesity, infertility and diabetes in a variety of mouse phenotypes. The demonstration that OB protein (also known as leptin) can normalize body weight in ob/ob mice has generated enormous interest. Most human obesity does not appear to result from a mutant form of leptin: rather, serum leptin concentrations are increased and there is an apparent inability to transport it to the central nervous system (CNS). Injection of leptin into the CNS of overfed rodents resistant to peripheral administration was found to induce biological activity. Consequently, for the leptin to act as a weight-lowering hormone in human obesity, it appears that appropriate concentrations must be present in the CNS. This places a premium on understanding the structure of the hormone in order to design more potent and selective agonists. Here we report the crystal structure at 2.4A resolution of a human mutant OB protein (leptin-E100) that has comparable biological activity to wild type but which crystallizes more readily. The structure reveals a four-helix bundle similar to that of the long-chain helical cytokine family.

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Advances in experimental and computational techniques have reaffirmed the role of protein X-ray crystallography as one of the primary providers of structural information both to enhance our fundamental understanding of biological systems and also to assist the design and optimization of important therapeutics. Today, the most important challenge facing macromolecular X-ray crystallography is the need to grow suitable crystals of a given protein. Once this has been accomplished, most often the question is not whether the structure will be solved but rather how fast this will be done. A dramatic example of this is the crystal structure of cytochrome c oxidase. The search for crystallization conditions took about 15 years and then the structure was solved in about one year.

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Phospholipases (PLAs) produce rate-limiting precursors in the biosynthesis of various types of biologically active lipids involved in inflammatory processes. Increased levels of human nonpancreatic secretory phospholipase A2 (hnps-PLA2) have been detected in several pathological conditions. An inhibitor of this enzyme could have therapeutic utility. A broad screening program was carried out to identify chemical structures which could inhibit hnps-PLA2. One of the lead compounds generated by the screening program was 5-methoxy-2-methyl-1-(phenylmethyl)-1H-indole-3-acetic acid (13a). We describe the syntheses, structure--activity relationships, and pharmacological activities of a series of indole-3-acetamides and related compounds derived from this lead. This SAR was undertaken with the aid of X-ray crystal structures of complexes between the inhibitors and hnps-PLA2 which were of great value in directing the SAR.

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As reported in our previous paper, a series of indole-3-acetamides which possessed potency and selectivity as inhibitors of human nonpancreatic secretory phospholipase A2(hnps-PLA2) was developed. The design of these compounds was based on information derived from x-ray crystal structures determined for complexes between the enzyme and its inhibitors. We describe here the further implementation of this structure-based design strategy and continued SAR development to produce indole-3-acetamides with additional functionalities which provide increased interaction with important residues within the enzyme active site. These efforts led to inhibitors with substantially enhanced potency and selectivity.

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The preceding papers of this series detail the development of functionalized indole-3-acetamides as inhibitors of hnps-PLA2. We describe here the extension of the structure-activity relationship to include a series of indole-3-glyoxamide derivatives. Functionalized indole-3-glyoxamides with an acidic substituent appended to the 4- or 5-position of the indole ring were prepared and tested as inhibitors of hnps-PLA2. It was found that the indole-3-glyoxamides with a 4-oxyacetic acid substituent had optimal inhibitory activity. These inhibitors exhibited an improvement in potency over the best of the indole-3-acetamides, and LY315920 (6m) was selected for evaluation clinically as an hnps-PLA2 inhibitor.

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A lead compound obtained from a high volume human non-pancreatic secretory phospholipase A2 (hnps-PLA2) screen has been developed into a potent inhibitor using detailed structural knowledge of inhibitor binding to the enzyme active site. Four crystal structures of hnps-PLA2 complexed with a series of increasingly potent indole inhibitors were determined and used as the structural basis for both understanding this binding and providing valuable insights for further development. The application of structure-based drug design has made possible improvements in the binding of this screening lead to the enzyme by nearly three orders of magnitude. Furthermore, the optimized structure (LY311727) displayed 1,500-fold selectivity when assayed against porcine pancreatic s-PLA2.

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Phospholipases A2 (PLA2s) may be grouped into distinct families of proteins that catalyse the hydrolysis of the 2-acyl bond of phospholipids and perform a variety of biological functions. The best characterized are the small (relative molecular mass approximately 14,000) calcium-dependent, secretory enzymes of diverse origin, such as pancreatic and venom PLA2s. The structures and functions of several PLA2s are known. Recently, high-resolution crystal structures of complexes of secretory PLA2s with phosphonate phospholipid analogues have provided information about the detailed stereochemistry of transition-state binding, confirming the proposed catalytic mechanism of esterolysis. By contrast, studies on mammalian nonpancreatic secretory PLA2s (s-PLA2s) have only recently begun; s-PLA2s are scarce in normal cells and tissues but large amounts are found in association with local and systemic inflammatory processes and tissue injury in animals and man. Such s-PLAs have been purified from rabbit and rat inflammatory exudate, from synovial fluid from patients with rheumatoid arthritis and from human platelets. Cloning and sequencing shows that the primary structure of the human s-PLA2 has about 37% homology with that of bovine pancreatic PLA2 and 44% homology with that of Crotalus atrox PLA2. The human s-PLA2 is an unusually basic protein, yet contains most of the highly conserved amino-acid residues and sequences characteristic of the PLA2s sequenced so far. Here we report the refined, three-dimensional crystal structure at 2.2 A resolution of recombinant human rheumatoid arthritic synovial fluid PLA2. This may aid the development of potent and specific inhibitors of this enzyme using structure-based design.

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The crystal structure of the trp repressor/operator complex shows an extensive contact surface, including 24 direct and 6 solvent-mediated hydrogen bonds to the phosphate groups of the DNA. There are no direct hydrogen bonds or non-polar contacts to the bases that can explain the repressor's specificity for the operator sequence. Rather, the sequence seems to be recognized indirectly through its effects on the geometry of the phosphate backbone, which in turn permits the formation of a stable interface. Water-mediated polar contacts to the bases also appear to contribute part of the specificity.

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An orthorhombic crystal form of trp repressor (aporepressor plus L-tryptophan ligand) was solved by molecular replacement, refined to 1.65 A resolution, and compared to the structure of the repressor in trigonal crystals. Even though these two crystal forms of repressor were grown under identical conditions, the refined structures have distinctly different conformations of the DNA-binding domains. Unlike the repressor/aporepressor structural transition, the conformational shift is not caused by the binding or loss of the L-tryptophan ligand. We conclude that while L-tryptophan binding is essential for forming a specific complex with trp operator DNA, the corepressor ligand does not lock the repressor into a single conformation that is complementary to the operator. This flexibility may be required by the various binding modes proposed for trp repressor in its search for and adherence to its three different operator sites.

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Comparison of the crystal structure of inactive unliganded trp aporepressor with that of trp repressor shows that binding tryptophan activates the dimer a thousandfold by moving two symmetrically-disposed flexible bihelical motifs. These flexible 'DNA-reading heads' flank a highly inflexible core domain formed by an unusual arrangement of interlocking alpha-helices from both subunits.

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Crystals of a simulated trp repressor-operator complex have been grown that are large enough and are sufficiently well ordered and durable to provide a high quality molecular image of this regulatory protein X DNA complex to better than 3-A resolution. The "operator" consists of a 2-fold rotationally symmetric 18-base pair duplex that is extended by a dT residue at both 5'-termini. This system exhibits extensive crystal polymorphism. The crystal form and diffraction properties are very sensitive to the length and terminal structure of the operator fragment, as well as the type and concentration of multivalent ions. When combined with the experience reported by others, our results do not support a consistent strategy for crystallization of protein X DNA complexes.

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The crystal structure of the Escherichia coli trp repressor has been solved to atomic resolution. The dimeric protein has a remarkable subunit interface in which five of each subunit's six helices are interlinked. The binding of L-tryptophan activates the aporepressor indirectly by fixing the orientation of the second helix of the helix-turn-helix motif and by moulding the details of the repressor's structure near the DNA binding surface.

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We have reproducibly grown crystals of L-tryptophan . trp aporepressor and indole-3-propionate . trp aporepressor complexes from Escherichia coli which are suitable for x-ray diffraction analysis. The active repressor, L-tryptophan . aporepressor, crystallizes in both trigonal (P3(1)21 or P3(2)21) and tetragonal (P4(1)22 or P4(3)22) forms which diffract to at least 2.0 and 2.5 A, respectively. The trigonal form has one-half of the functional dimer/asymmetric unit; therefore, the trp repressor molecule has an axis of 2-fold rotational symmetry corresponding to the lattice dyad. The inactive complex, indole-3-propionate . aporepressor, or "pseudorepressor," forms tetragonal crystals that also diffract to at least 2.5 A and are isomorphous to those of the active repressor. Slight differences between their diffraction patterns indicate modest structural differences between active and inactive complexes that are presumably mediated by the alpha-amino group of L-tryptophan and account for operator-specific binding.

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An isomorphous osmium derivative of crystalline yeast initiator transfer RNA has been prepared and interpreted to 6-angstrom resolution. The coordinates of the heavy atoms have been determined by Patterson and "direct" methods applied to the difference coefficients of the centric projections, followed by least-squares refinement. There is one dominant site per asymmetric unit, consistent with the finding by neutron-activation analysis that there is approximately one osmium atom per molecule of transfer RNA. The osmium derivative appears to be a normal substrate for enzymatic aminoacylation.

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