Adriamycin-Induced Models of VACTERL Association.

Animal models are of great importance for medical research. They have enabled analysis of the aetiology and pathogenesis of complex congenital malformations and have also led to major advances in the surgical and therapeutic management of these conditions. Animal models allow us to comprehend the morphological and molecular basis of disease and consequently to discover novel approaches for both surgical and medical therapy. The anthracycline antibiotic adriamycin was incidentally found to have teratogenic effects on rats, producing a range of defects remarkably similar to the VACTERL association of congenital anomalies in humans, providing a reproducible animal model of this condition. VACTERL association is a spectrum of birth defects which includes vertebral, anal, cardiovascular, tracheo-oesophageal, renal and limb anomalies. In recent years, adriamycin rodent models of VACTERL have provided valuable insights into the pathogenesis of this complex association, particularly in relation to tracheo-oesophageal malformations. The adriamycin rat model and adriamycin mouse model are now well established in the investigation of the morphology of faulty organogenesis and the regulation of gene expression in tracheo-oesophageal anomalies.

Rational design of diflunisal analogues with reduced affinity for human serum albumin.

Many lead compounds bind to serum albumin and exhibit markedly reduced efficacy in vivo as compared to their potency in vitro. To aid in the design of compounds with reduced albumin binding, we performed nuclear magnetic resonance (NMR) structural and binding studies on the complex between domain III of human serum albumin (HSA-III) and diflunisal, a cyclooxygenase inhibitor with antiinflammatory activity. The structural studies indicate that the aromatic rings of diflunisal are involved in extensive and specific interactions with hydrophobic residues that comprise the binding pocket in subdomain IIIA. The carboxylic acid of diflunisal forms electrostatic interactions with the protein similar to those observed in the X-ray structure of HSA complexed to myristic acid. In addition to the structural studies, NMR-derived binding constants were obtained for diflunisal and closely related analogues to develop a structure-affinity relationship for binding to subdomain IIIA. On the basis of the structural and binding data, compounds were synthesized that exhibit more than a 100-fold reduction in binding to domain III of HSA, and nearly a 10-fold reduction in affinity for full length albumin. Significantly, several of these compounds maintain activity against cyclooxygenase-2. These results suggest a rational strategy for designing out albumin binding in potential drug molecules by using structure-based design in conjunction with NMR-based screening.

Structure of the N-terminal region of Haemophilus influenzae H10017: implications for function.

Haemophilus influenzae is a gram-negative pathogen that causes infections ranging from asymptomatic colonization of the human upper respiratory tract to serious invasive diseases such as meningitis. Although the genome of Haemophilus influenzae has been completely sequenced, the structure and function of many of these proteins are unknown. H10017 is one of these uncharacterized proteins. Here we describe the three-dimensional solution structure of the N-terminal portion of H10017 as determined by NMR spectroscopy. The structure consists of a five-stranded antiparallel beta-sheet and two short alpha-helices. It is similar to the C-terminal domain of Diphtheria toxin repressor (DtxR). The C-terminal portion of H10017 has an amino acid sequence that closely resembles pyruvate formate-lyase--an enzyme that converts pyruvate and CoA into acetyl-CoA and formate by a radical mechanism. Based on structural and sequence comparisons, we propose that the C-terminus of H10017 functions as an enzyme with a glycyl radical mechanism, while the N-terminus participates in protein/protein interactions involving an activase (iron-sulfur protein) and/or the substrate.

Design of adenosine kinase inhibitors from the NMR-based screening of fragments.

A strategy is described for designing high-affinity ligands using information derived from the NMR-based screening of fragments. The method involves the fragmentation of an existing lead molecule, identification of suitable replacements for the fragments, and incorporation of the newly identified fragments into the original scaffold. Using this technique, novel substituents were rapidly identified and incorporated into lead inhibitors of adenosine kinase that exhibited potent in vitro and in vivo activities. This approach is a valuable strategy for modifying existing leads to improve their potency, bioavailability, or toxicity profile and thus represents a useful technique for lead optimization.

Identification of novel inhibitors of urokinase via NMR-based screening.

Using an NMR-based screen, a novel class of urokinase inhibitors were identified that contain a 2-aminobenzimidazole moiety. The inhibitory potency of this family of inhibitors is similar to that of inhibitors containing a guanidine or amidine group. However, unlike previously described guanidino- or amidino-based inhibitors which have pK(a) values greater than 9.0, urokinase inhibitors containing a 2-aminobenzimidazole have pK(a) values of 7.5. Thus, 2-aminobenzimidazoles may have improved pharmacokinetic properties which could increase the bioavailability of inhibitors which contain this moiety. A crystal structure of one of the lead inhibitors, 2-amino-5-hydroxybenzimidazole, complexed with urokinase reveals the electrostatic and hydrophobic interactions that stabilize complex formation and suggests nearby subsites that may be accessed to increase the potency of this new series of urokinase inhibitors.

Privileged molecules for protein binding identified from NMR-based screening.

A statistical analysis of NMR-derived binding data on 11 protein targets was performed to identify molecular motifs that are preferred for protein binding. The analysis indicates that compounds which contain a biphenyl substructure preferentially bind to a wide range of proteins and that high levels of specificity (>250-fold) can be achieved even for these small molecules. These results suggest that high-throughput screening libraries that are enriched with biphenyl-containing compounds can be expected to have increased chances of yielding high-affinity ligands for proteins, and they suggest that the biphenyl can be utilized as a template for the discovery and design of therapeutics with high affinity and specificity for a broad range of protein targets.

Novel inhibitors of Erm methyltransferases from NMR and parallel synthesis.

The Erm family of methyltransferases confers resistance to the macrolide-lincosamide-streptogramin type B (MLS) antibiotics through the methylation of 23S ribosomal RNA. Upon the methylation of RNA, the MLS antibiotics lose their ability to bind to the ribosome and exhibit their antibiotic activity. Using an NMR-based screen, we identified a series of triazine-containing compounds that bind weakly to ErmAM. These initial lead compounds were optimized by the parallel synthesis of a large number of analogues, resulting in compounds which inhibit the Erm-mediated methylation of rRNA in the low micromolar range. NMR and X-ray structures of enzyme/inhibitor complexes reveal that the inhibitors bind to the S-adenosylmethionine binding site on the Erm protein. These compounds represent novel methyltransferase inhibitors that serve as new leads for the reversal of Erm-mediated MLS antibiotic resistance.

NMR-based discovery of phosphotyrosine mimetics that bind to the Lck SH2 domain.

Using an NMR-based screen, a series of novel phosphotyrosine mimetics were discovered that bind to the SH2 domain of Lck. These compounds may serve as useful leads for the design of nonpeptide inhibitors of SH2 domains with improved bioavailability and metabolic stability compared to the natural ligands that contain phosphotyrosine.

High-throughput nuclear magnetic resonance-based screening.

A high-throughput screening strategy is described that involves the acquisition of two-dimensional 15N/1H correlation spectra in less than 10 min on 50 microM protein samples using cryogenic NMR probe technology. By screening at these concentrations, small organic molecules can be tested in mixtures of 100, which dramatically increases the throughput of the NMR-based assay. Using this strategy, libraries of more than 200 000 compounds can be tested in less than 1 month. There are many advantages of high-throughput NMR-based screening compared to conventional assays, such as the ability to identify high-affinity ligands for protein targets with no known function. This suggests that the method will be extremely useful for screening the large number of targets derived from genomics research.

Evaluation of PMF scoring in docking weak ligands to the FK506 binding protein.

A new knowledge-based scoring function (PMF-score), implemented into the DOCK4 program, was used to screen a database of 3247 small molecules for binding to the FK506 binding protein (FKBP). The computational ranking of these compounds was compared to the binding affinities measured by NMR. It was demonstrated that small, weakly binding molecules have, on average, higher computational scores than nonbinders and are enriched in the upper ranks of the computational scoring lists. In addition, the results obtained with the PMF scoring function were superior (by 30-120% larger enrichment factors) to those obtained with the standard force field score of DOCK4. The reliable ranking of small, weakly binding molecules offers new ways of designing building blocks in combinatorial libraries as well as SAR by NMR libraries with the increased chance of identifying suitable lead compounds for drug design.

NMR-based discovery of lead inhibitors that block DNA binding of the human papillomavirus E2 protein.

The E2 protein is required for the replication of human papillomaviruses (HPVs), which are responsible for anogenital warts and cervical carcinomas. Using an NMR-based screen, we tested compounds for binding to the DNA-binding domain of the HPV-E2 protein. Three classes of compounds were identified which bound to two distinct sites on the protein. Biphenyl and biphenyl ether compounds containing a carboxylic acid bind to a site near the DNA recognition helix and inhibit the binding of E2 to DNA. Benzophenone-containing compounds which lack a carboxylic acid group bind to the beta-barrel formed by the dimer interface and exhibit negligible effects on the binding of E2 to DNA. Structure-activity relationships from the biphenyl and biphenyl ether compounds were combined to produce a compound [5-(3'-(3",5"-dichlorophenoxy)-phenyl)-2,4-pentadienoic acid] with an IC50 value of approximately 10 microM. This compound represents a useful lead for the development of antiviral agents that interfere with HPV replication and further illustrates the usefulness of the SAR by NMR method in the drug discovery process.

Solution dynamics of the 1,2,3,4,6-penta-O-acetyl-alpha-D-idopyranose ring.

The anticoagulant properties of heparin are thought to derive from the inhibition of thrombin and other coagulation-related proteases by the binding of heparin to cofactors such as antithrombin III and heparin cofactor II. The apparent minimum native heparin sequence which can bind to antithrombin III is a highly sulfated pentasaccharide which contains a 2-O-sulfo-alpha-L-idopyranosyluronic acid residue. The idopyranosyl residue has the unusual property of existing in the solution state as a mixture of ring conformers. Whereas most hexopyranose sugars exist as a single chair conformer (eg D-glucose exists overwhelmingly as a (4)C1 chair), the idopyranosyl ring is known to rapidly exchange between at least two and often more distinct conformations, depending on type and number of substituents (hydroxyl, carboxyl, sulfate, etc.) and solvent conditions (solvent pH, salt concentration, temperature). It is believed that this flexibility of the idopyranosyl residue in heparin is related to its binding specificity. In the past, coupling constants and molecular dynamics have been used to estimate the relative populations of conformers in iduronate and related compounds. Here we report extensive NMR measurements, including line shape analysis, T1p measurements, T1 and NOE measurements and spectral density mapping, which have been used to study the dynamics of conformer interconversion in model compounds related to idose and glucose. The findings presented here indicate that 1,2,3,4,6-penta-O-acetyl-alpha-D-idopyranose can be reasonably well described as existing in a two-state equilibrium consisting of the (4)C1 and (0)S2 conformers. (13)C NMR line shape analysis yields a deltaH+ of 40 kJ mol(-1) and a deltaS++ of 31 J mol(-1) K(-1) for the (4)C1 --> (0)S2 interconversion and a deltaH++ of 31 kJ mol(-1) and a deltaS++ of 13 J mol(-1) K(-1) for the (0)S2 --> (4)C1 interconversion. This corresponds to exchange rates of 22 and 128 MHz, respectively, at room temperature.

Discovering high-affinity ligands for proteins: SAR by NMR.

A nuclear magnetic resonance (NMR)-based method is described in which small organic molecules that bind to proximal subsites of a protein are identified, optimized, and linked together to produce high-affinity ligands. The approach is called "SAR by NMR" because structure-activity relationships (SAR) are obtained from NMR. With this technique, compounds with nanomolar affinities for the FK506 binding protein were rapidly discovered by tethering two ligands with micromolar affinities. The method reduces the amount of chemical synthesis and time required for the discovery of high-affinity ligands and appears particularly useful in target-directed drug research.

Structural characterization of the interaction between a pleckstrin homology domain and phosphatidylinositol 4,5-bisphosphate.

The pleckstrin homology (PH) domain is a protein module of approximately 100 amino acids that is found in several proteins involved in signal transduction [for a recent review, see Gibson et al. (1994) Trends Biochem. Sci. 19, 349-353]. Although the specific function of the PH domain has not yet been elucidated, many of the proteins which contain this domain associate with phospholipid membranes, and PH domains have been shown to bind to phosphatidylinositol 4,5-bisphosphate (PIP2) [Harlan et al. (1994) Nature 371, 168-170] and the beta gamma subunits of G-proteins [Touhara et al. (1994) J. Biol. Chem. 269, 10217-10220]. We have postulated that pleckstrin homology domains may be important for the translocation of proteins to the membrane by an interaction with the negatively charged head group of phospholipids. Here we show the importance of three conserved lysine residues for binding to PIP2 by site-directed mutagenesis. These results should aid future site-directed mutagenesis studies in probing the function of PIP2-PH domain interactions in the various proteins containing this module. In addition, we examine the specificity of this binding and illustrate the importance of charge--charge interactions in PIP2-PH domain complex formation from binding experiments involving PIP2 analogs.

A genetic algorithm-based protocol for docking ensembles of small ligands using experimental restraints.

A genetic algorithm (GA) based method for docking ensembles of small, flexible ligands to receptor proteins using NMR-derived constraints is described. In this method, three translations and rotations of the ligand and the dihedral angles of the ligand are represented by binary strings and evolve under the genetic operators of cross-over, mutation, migration and selection. The fitness function for the selection process includes distance and dihedral restraints and a repulsive van der Waals term. The GA was applied to a three-atom model system as well as to the streptavidin-biotin complex using simulated intermolecular distance restraints. In both systems, the GA was able to obtain low-energy conformations when only a single binding site was simulated. Calculations were also performed using distance restraints from two distinct binding sites. In these simulations, the GA was able to obtain low-energy conformations corresponding to ligand molecules in each of the two sites. The inclusion of additional ligands in the ensemble did not result in an energetic benefit, confirming that only two ligand conformations were necessary to fulfill the distance restraints. This method allows for a direct investigation of the minimum number of ligand orientations necessary to fulfill experimental distance restraints, and simultaneously yields detailed structural information about each site.

Pleckstrin homology domains bind to phosphatidylinositol-4,5-bisphosphate.

The pleckstrin homology (PH) domain is a new protein module of around 100 amino acids found in several proteins involved in signal transduction. Although its specific function has yet to be elucidated, the carboxy-terminal regions of many PH domains bind to the beta gamma subunits of G proteins. On the basis of structural similarities between PH domains and lipid-binding proteins, we have proposed that PH domains may be binding to lipophilic molecules. Indeed, many of the proteins that contain this domain associate with phospholipid membranes, and disruption of this domain can interfere with membrane association. Here we report that PH domains bind to phosphatidylinositol-4,5-bisphosphate and show that the lipid-binding site is located at the lip of the beta-barrel. This suggests that PH domains may be important for membrane localization of proteins through interactions with phosphatidylinositol-4,5-bisphosphate.

Solution structure of a pleckstrin-homology domain.

Pleckstrin, the major protein kinase C substrate of platelets, contains domains of about 100 amino acids at the amino and carboxy termini that have been found in a number of proteins, including serine/threonine kinases, GTPase-activating proteins, phospholipases and cytoskeletal proteins. These conserved sequences, termed pleckstrin-homology (PH) domains, are thought to be involved in signal transduction. But the details of the function and binding partners of the PH domains have not been characterized. Here we report the solution structure of the N-terminal pleckstrin-homology domain of pleckstrin determined using heteronuclear three-dimensional nuclear magnetic resonance spectroscopy. The structure consists of an up-and-down beta-barrel of seven antiparallel beta-strands and a C-terminal amphiphilic alpha-helix that caps one end of the barrel. The overall topology of the domain is similar to that of the retinol-binding protein family of structures.