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Studies the discovery of potent nonpeptide inhibitors of stromelysin by the structure-activity relationships by nuclear magnetic resonance (SAR by NMR). Description of the SAR by NMR process; Identification of ligands which bind weakly to stromelysin; Minimal requirement of chemical synthesis.

Activating mutations in KRAS are the most frequent oncogenic alterations in cancer. The oncogenic hotspot position 12, located at the lip of the switch II pocket, offers a covalent attachment point for KRAS G12C inhibitors. To date, KRAS G12C inhibitors have been discovered by first covalently binding to the cysteine at position 12 and then optimizing pocket binding. We report on the discovery of the in vivo active KRAS G12C inhibitor BI-0474 using a different approach, in which small molecules that bind reversibly to the switch II pocket were identified and then optimized for non-covalent binding using structure-based design. Finally, the Michael acceptor containing warhead was attached. Our approach offers not only an alternative approach to discovering KRAS G12C inhibitors but also provides a starting point for the discovery of inhibitors against other oncogenic KRAS mutants.

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.

The interaction between leukocyte function-associated antigen-1 (LFA-1) and intracellular adhesion molecule-1 (ICAM-1) has been implicated in inflammatory and immune diseases. Recently, a novel series of p-arylthio cinnamides has been described as potent antagonists of the LFA-1/ICAM-1 interaction. These compounds were found to bind to the I domain of LFA-1 using two-dimensional NMR spectroscopy of 15N-labeled LFA-1 I domain. On the basis of NOE studies between compound 1 and the I domain of LFA-1, a model of the complex was constructed. This model revealed that compound 1 does not directly inhibit ICAM-1 binding by interacting with the metal ion dependent adhesion site (MIDAS). Instead, it binds to the previously proposed I domain allosteric site (IDAS) of LFA-1 and likely modulates the activation of LFA-1 through its interaction with this regulatory site. A fragment-based NMR screening strategy was applied to identify small, more water-soluble ligands that bind to a specific region of the IDAS. When incorporated into the parent cinnamide template, the resulting analogues exhibited increased aqueous solubility and improved pharmacokinetic profiles in rats, demonstrating the power of this NMR-based screening approach for rapidly modifying high-affinity ligands.

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.

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.

The Bcl-2 family of proteins play a pivotal role in the regulation of programmed cell death. One of the postulated mechanisms for the function of these proteins involves the formation of ion channels in membranes. As a first step to structurally characterize these proteins in a membrane environment, we investigated the structure of a Bcl-x(L) mutant protein when incorporated into small detergent micelles. This form of Bcl-x(L) lacks the loop (residues 49-88) between helix 1 and helix 2 and the putative C-terminal transmembrane helix (residues 214-237). Below the critical micelle concentration (CMC), Bcl-x(L) binds detergents in the hydrophobic groove that binds to pro-apoptotic proteins. However, above the CMC, Bcl-x(L) undergoes a dramatic conformational change. Using NMR methods, we characterized the secondary structure of Bcl-x(L) in the micelle-bound form. Like Bcl-x(L) in aqueous solution, the structure of the protein when dissolved in dodecylphosphocholine (DPC) micelles consists of several alpha-helices separated by loops. However, the length and position of the individual helices of Bcl-x(L) in micelles differ from those in aqueous solution. The location of Bcl-x(L) within the micelle was examined from the analysis of protein-detergent NOEs and limited proteolysis. In addition, the mobility of the micelle-bound form of Bcl-x(L) was investigated from NMR relaxation measurements. On the basis of these studies, a model is proposed for the structure, dynamics, and location of Bcl-x(L) in micelles. In this model, Bcl-x(L) has a loosely packed, dynamic structure in micelles, with helices 1 and 6 and possibly helix 5 partially buried in the hydrophobic interior of the micelle. Other parts of the protein are located near the surface or on the outside of the micelle.

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.

C24-Deoxyascomycin was prepared in a two-step process from ascomycin and evaluated for its immunosuppressant activity relative to ascomycin and FK506. An intermediate in the synthetic pathway, Delta(23,24)-dehydroascomycin, was likewise evaluated. Despite lacking the hydrogen-bonding interactions associated with the C24-hydroxyl moiety of ascomycin, C24-deoxyascomycin was found to be equipotent to the parent compound both in its immunosuppressive potency and in its interaction with the immunophilin, FKBP12. Conversely, Delta(23,24)-dehydroascomycin which also lacks the same hydrogen-bonding interactions did not exhibit this potency. NMR studies were conducted on the FKBP12/C24-deoxyascomycin complex in an attempt to understand this phenomenon at the molecular level. The NMR structures of the complexes formed between FKBP12 and ascomcyin or C24-deoxyascomcyin were very similar, suggesting that hydrogen-bonding interactions with the C24 hydroxyl moiety are not important for complex formation.

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.

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.

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.

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.

Proteins recognize ligands by forming specific intermolecular interactions that often involve solvent exposed residues. Changes in the motional properties of these residues upon binding can affect the conformational entropy of the system and thus are related to the energetics of binding. The role that dynamics plays in ligand recognition can be investigated by comparing the motional properties of a free and ligated protein. NMR relaxation studies are well suited for examining changes in dynamics, especially for motions on a nanosecond to picosecond time scale. Recently, we determined the solution structure of the phosphotyrosine binding (PTB) domain of the insulin receptor substrate (IRS-1) complexed to a tyrosine-phosphorylated peptide derived from the interleukin 4 (IL-4) receptor [Zhou et al., (1996) Nat. Struct. Biol. 3, 388-393]. The peptide binds tightly to the protein in a surface exposed pocket, resulting in the partial burial of many protein residues. Using NMR relaxation studies, the dynamics of the backbone nitrogens of IRS-1 PTB domain were studied in both the free protein and the protein when complexed to the IL-4 receptor phosphopeptide. The backbone nitrogens of many residues that make important contacts to the ligand are motionally restricted in the free and complexed protein. Additional residues become motionally restricted only after ligand binding, including several residues that do not make any direct contacts with the ligand. These observed changes in the dynamics are compared to structural features of the complex.

The backbone dynamics of the C-terminal DNA-binding domain of Escherichia coli topoisomerase I has been characterized in the absence and presence of single-stranded DNA by NMR spectroscopy. 15N spin-lattice relaxation times (T1), spin-spin relaxation times (T2), and heteronuclear NOEs were determined for the uniformly 15N-labeled protein. These data were analyzed by using the model-free formalism to derive the model-free parameters (S2, tau e, and R(ex)) for each backbone N-H bond vector and the overall molecular rotational correlation time (tau m)., The molecular rotational correlation time tau m was determined to be 7.49 +/- 0.36 ns for the free and 12.7 +/- 1.07 ns for the complexed protein. Several residues were found to be much more mobile than the average, including 11 residues at the N-terminus, 2 residues at the C-terminus, and residues 25 and 31-35 which are located in a region of the protein that binds to DNA. The binding of ssDNA to the free protein causes a slight increase in the order parameters (S2) for a small number of residues and a slight decrease in the order parameters (S2) for the majority of the residues. In particular, upon binding to ssDNA, the mobility of the first alpha-helix and the two beta-sheets was slightly increased, and the mobility of a few specific residues in the loops/turns was restricted. These results differ from the previous studies on the backbone dynamics of molecular complexes in which reduced mobilities were typically observed upon ligand binding.

The three-dimensional structure of the DNA-binding domain of the E2 protein from human papillomavirus-31 was determined by using multidimensional heteronuclear nuclear magnetic resonance (NMR) spectroscopy. A total of 1429 NMR-derived distance and dihedral angle restraints were obtained for each of the 83-residue subunits of this symmetric dimer. The average root mean square deviations of 20 structures calculated using a distance geometry-simulated annealing protocol are 0.59 and 0.90 angstroms for the backbone and all heavy atoms, respectively, for residues 2-83. The structure of the human virus protein free in solution consists of an eight-stranded beta-barrel and two pairs of alpha-helices. Although the overall fold of the protein is similar to the crystal structure of the bovine papillomavirus-1 E2 protein when complexed to DNA, several small but interesting differences were observed between these two structures at the subunit interface. In addition, a beta-hairpin that contacts DNA in the crystal structure of the protein-DNA complex is disordered in the NMR structures, and steady-state 1H-15N heteronuclear NOE measurements indicate that this region is highly mobile in the absence of DNA. The recognition helix also appears to be flexible, as evidenced by fast amide exchange rates. This phenomenon has also been observed for a number of other DNA-binding proteins and may constitute a common theme in protein/DNA recognition.

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.

Escherichia coli DNA topoisomerase I catalyzes the interconversion of different topological forms of DNA. In this paper we describe NMR studies of a 14K C-terminal fragment of this enzyme that binds preferentially to single-stranded DNA and enhances the enzyme's ability to relax negatively supercoiled DNA under high salt conditions. The 1H, 13C, and 15N resonances of the protein were assigned from a number of heteronuclear multidimensional NMR experiments, and the three-dimensional structure of the protein was determined from a total of 2188 NMR-derived restraints. The root-mean-square deviation about the mean coordinate positions for residues 13-120 is 0.68 +/- 0.11 A for the backbone atoms and 1.09 +/- 0.09 A for all heavy atoms. The overall fold, which consists of two four-stranded beta-sheets separated by two helices, differs from other DNA- and RNA-binding proteins such as gene 5, cold shock protein, and hnRNP C. From an analysis of the changes in chemical shift upon the addition of single-stranded DNA, the location of the oligonucleotide binding site was determined. The binding site consists of a beta-sheet containing positively charged and aromatic amino acids and, in spite of its different structure, is similar to that found in other proteins that bind single-stranded oligonucleotides.

The amino-terminal fragment (ATF) of urokinase-type plasminogen activator (u-PA) is a two-domain protein which consists of a kringle and a growth factor domain (GFD). The dynamics of uniformly 15N-labeled ATF was examined by measuring the longitudinal (T1) and transverse (T2) 15N relaxation times and heteronuclear NOEs. The data were interpreted in terms of the model-independent spectral density function. The GFD was found to exhibit a high degree of anisotropy, whereas the kringle domain of ATF undergoes isotropic reorientation. This difference in anisotropy is best explained by the two domains moving independently such as differently shaped beads on a string. With the exception of the N- and C-terminal regions of the protein, the most flexible region of ATF was the seven-residue omega loop (N22-I28) of the GFD which has been implicated in the binding of u-PA to its receptor. The amides of the linker region between the domains displayed high values of the order parameter, indicating restricted motion on the picosecond time scale. This is in contrast to the flexible linker of calmodulin [Barbato et al. (1992) Biochemistry 31, 5269-5278], which displayed low values of S2 and unrestricted motion in the linker region.

Protein tyrosine phosphatases (PTPs) are important enzymes involved in signal transduction, cell cycle regulation, and the control of differentiation. Despite the importance of this class of enzymes in the control of critical cell processes, very little structural information is available for this family of proteins. In this paper, we present the first solution structure of a protein tyrosine phosphatase. This protein is a low molecular weight cytosolic PTP that was initially isolated from bovine heart. The structure that was determined from 1747 NMR-derived restraints consists of a central four-stranded parallel beta-sheet surrounded by four alpha-helices and a short 3(10) helix. The phosphate binding site, identified by chemical shift changes upon the addition of the competitive inhibitors phosphate and vanadate, is in a loop region connecting the C-terminal end of the first beta-strand with the first alpha-helix. Residues in the second, fourth, and fifth alpha-helices and in some of the loop regions connecting the elements of regular secondary structure also contribute to the binding site. The structure determined here is consistent with previous mutagenesis and chemical modification studies conducted on this protein.

Phosphotyrosyl protein phosphatases play an important role in mediating cellular signal transduction; yet three-dimensional structures of this important class of proteins have not been reported. We present the sequence-specific 1H, 13C, and 15N backbone assignments for the low molecular weight bovine heart phosphotyrosyl protein phosphatase (BHPTPase) (157 residues, 17,900). The assignments were obtained from a combination of double- and triple-resonance multidimensional NMR experiments. From these assignments, the secondary structure of BHPTPase was determined from an analysis of NOE patterns, 3JHNH alpha coupling constants, 13C alpha and 13CO chemical shifts, and amide 1H exchange rates. BHPTPase was found to consist of a four-stranded parallel beta-sheet (residues K6-C12, W39-A45, Y87-M91, and K112-L116), four alpha-helices (residues I21-D32, R58-G67, S94-N104, and D135-R157), and one stretch of beta 10-helix (residues K79-F85). The secondary structure is characteristic of the beta alpha beta structural motif. The secondary structure elements identified in this study are consistent with previous chemical and mutagenesis studies of BHPTPase structure.

The amino-terminal fragment (ATF) of urokinase-type plasminogen activator is a two domain protein which consists of a growth factor and a kringle domain. The 1H, 13C, and 15N chemical shifts of this protein have been assigned using heteronuclear two- and three-dimensional NMR experiments on selective and uniformly 15N- and 15N/13C-labeled protein isolated from mammalian cells that overexpress the protein. The chemical shift assignments were used to interpret the NOE data which resulted in a total of 1299 NOE restraints. The NOE restraints were used along with 27 phi angle restraints and 21 hydrogen-bonding restraints to produce 15 low energy structures. The individual domains in the structures are highly converged, but the two domains are structurally independent. The root mean square deviations (rmsd) between residues 11-46 in the growth factor domain and the mean atomic coordinates were 0.99 +/- 0.2 for backbone heavy atoms and 1.65 +/- 0.2 for all non-hydrogen atoms. For residues 55-130 in the kringle domain, the rmsd was 0.84 +/- 0.2 for backbone heavy atoms and 1.42 +/- 0.2 for all non-hydrogen atoms. The overall structures of the individual domains are very similar to the structures of homologous proteins. However, important structural differences between the growth factor and other homologous proteins were observed in the region which has been implicated in binding the urokinase receptor which may explain, in part, why other growth factors show no appreciable affinity for the urokinase receptor.

From a series of 15N-resolved 3D ROESY-HMQC and 13C-resolved 3D NOESY-HMQC spectra of the FK506 binding protein (FKBP)/ascomycin complex in H2O, the locations of three tightly bound water molecules were identified. These waters are all buried within the interior of the complex and form an integral part of its structure via a network of hydrogen bonds. Water molecules in identical locations exhibiting a similar hydrogen bonding pattern were also observed in the X-ray crystal structures of FKBP/FK506 [Van Duyne, G. D., Standaert, R. F., Karplus, P. A., Schreiber, S. L., & Clardy, J. (1991) Science 252, 839-842] and FKBP/rapamycin [Van Duyne, G. D., Standaert, R. F., Schreiber, S. L., & Clardy, J. (1991) J. Am. Chem. Soc. 113, 7433-7434]. However, none of the surface waters observed in the X-ray structures were detected in the NMR experiments due to their fast exchange with bulk water. In order to examine the effects of the three internal water molecules on NMR structure determinations of the FKBP/ascomycin complex, two sets of NMR structures were calculated either with or without the waters. By including the three internal waters in the structure calculations, a decrease in the root mean square deviation and improved angular order parameters was observed for FKBP residues in the vicinity of the water molecules. In addition, subtle conformational differences were observed between NMR structures generated either with or without the waters.(ABSTRACT TRUNCATED AT 250 WORDS)

The equilibrium folding behavior of recombinant human FK-binding protein, a peptidyl-prolyl cis-trans-isomerase, was examined by urea-induced denaturation using probes of protein structure including intrinsic tryptophan fluorescence, second-derivative UV absorbance, CD, and NMR. All optical probes of protein structure indicate that FKBP is capable of folding reversibly. The second-derivative UV absorbance and CD probes of the structure exhibited urea denaturation transitions at approximately 4.3 M urea. The fluorescence of the single protein tryptophan is quenched in the folded state. During the unfolding-folding transition, the unquenching of tryptophan fluorescence occurs at a slightly lower urea concentration (3.9 M urea) than the changes observed for the other optical probes of folding. These probes of structure demonstrate little dependence on protein concentration in the range of 0.2- approximately 3 mg/mL across the urea-induced denaturation transition. The reversibility of the unfolding-folding transition was confirmed from two-dimensional 15N/1H heteronuclear single-quantum coherence (HSQC) spectra of [U-15N]FKBP. In addition, the native-denatured transitions for 57 individual amino acids were determined from an analysis of these spectra acquired at different urea concentrations. Analysis of the transitions for all clearly observable HSQC cross peaks for residues distributed throughout the protein and comparison to the optical folding transitions, indicate that FKBP global folding is consistent with a two-state process. Although direct measurement of FKBP catalytic activity in urea was complex, enzyme activity was observed up to the beginning of the FKBP urea-denaturation transition.(ABSTRACT TRUNCATED AT 250 WORDS)

A high-resolution three-dimensional solution structure of the FKBP/ascomycin complex has been determined using heteronuclear multidimensional nuclear magnetic resonance spectroscopy (NMR) and a distance geometry/simulated annealing protocol. A total of 43 structures of the complex, including 3 tightly bound water molecules, were obtained using 1958 experimental restraints consisting of 1724 nuclear Overhauser effect (NOE) derived distances, 66 chi 1 and 46 phi angular restraints, and 122 hydrogen bond restraints. The root mean square (rms) deviations between the 43 FKBP/ascomycin solution structures and the mean atomic coordinates were 0.43 +/- 0.08 A for the backbone heavy atoms and 0.80 +/- 0.08 A for all non-hydrogen atoms. Angular order parameters for the family of 43 conformations were calculated to determine dihedral convergence. Order parameters for phi, psi, and chi 1 angles exhibited mean values of 0.98, 0.97, and 0.95, respectively, while the mean of the chi 2 order parameter was 0.63. Comparisons were made between the FKBP/ascomycin complex and two NMR-derived solution structures of unbound FKBP and the X-ray crystal structure of an FKBP/FK506 complex. Differences were observed between the FKBP/ascomycin complex and uncomplexed FKBP for residues 33-45 and 78-92. In contrast, the NMR-derived solution structure of the FKBP/ascomycin complex and the X-ray crystal structure of the FKBP/FK506 complex were very similar. Differences between the two complexes were mainly observed in the conformations of some highly solvent exposed side chains.

A method to obtain uniformly isotopically labeled (15N and 15N/13C) protein from mammalian cells is described. The method involves preparation of isotopically labeled media consisting of amino acids isolated from bacterial and algal extracts supplemented with cysteine and enzymatically synthesized glutamine. The approach is demonstrated by producing 15N-labeled and 15N/13C-labeled urokinase from Sp2/0 cells and successfully growing Chinese hamster ovary (CHO) cells on the labeled media. Thus, using the procedures described, isotopically labeled proteins that have been expressed in mammalian cells can be prepared, allowing them to be studied by heteronuclear multidimensional NMR techniques.

Multidimensional, heteronuclear NMR methods were used to determine the complete 1H and 13C resonance assignments for [U-13C]ascomycin bound to recombinant FKBP, including stereospecific assignment of all 22 methylene protons. The conformation of ascomycin was then determined from an analysis of NOEs observed in a 13C-edited 3D HMQC-NOESY spectrum of the [U-13C]ascomycin/FKBP. This structure is found to be quite different from the solution structure of the two forms of uncomplexed FK-506. However, it is very similar to the X-ray crystal structure of FK-506 bound to FKBP, rms deviation = 0.56 A. The methods used for resonance assignment and structure calculation are presented in detail. Furthermore, FKBP/ascomycin NOEs are reported which help define the structure of the ascomycin binding pocket. This structural information obtained in solution was compared to the recently described X-ray crystal structure of the FKBP/FK-506 complex.

Cyclosporin A (CsA), a potent immunosuppressant, is known to bind with high specificity to cyclophilin (CyP), a 17.7 kDa protein with peptidyl-prolyl isomerase activity. In order to investigate the three-dimensional structure of the CsA/CyP complex, we have applied a variety of multidimensional NMR methods in the study of uniformly 13C-labeled CsA bound to cyclophilin. The 1H and 13C NMR signals of cyclosporin A in the bound state have been assigned, and from a quantitative interpretation of the 3D NOE data, the bound conformation of CsA has been determined. Three-dimensional structures of CsA calculated from the NOE data by using a distance geometry/simulated appealing protocol were found to be very different from previously determined crystalline and solution conformations of uncomplexed CsA. In addition, from CsA/CyP NOEs, the portions of CsA that interact with cyclophilin were identified. For the most part, those CsA residues with NOEs to cyclophilin were the same residues important for cyclophilin binding and immunosuppressive activity as determined from structure/activity relationships. The structural information derived in this study together with the known structure/activity relationships for CsA analogues may prove useful in the design of improved immunosuppressants. Moreover, the approach that is described for obtaining the structural information is widely applicable to the study of small molecule/large molecule interactions.

A series of structurally related pregnane analogues which exhibit a wide range of anesthetic potencies were incorporated into unilamellar egg lecithin vesicles and their relative mobilities examined with 1H and 2H high-resolution NMR spectroscopy. The data from this study reveal a trend suggesting a relationship between the motional properties of a steroid and its anesthetic potency. The data are congruent with the idea that anesthetic activity is associated with perturbation of the membrane bilayer by the steroid molecule; the degree to which the membrane is perturbed is apparently dependent upon the specific structural and stereochemical features of the steroid. This study supports the hypothesis that lipid bilayers are capable of a high degree of structural discrimination.

Molecular modeling methods have been used to design a novel series of conformationally constrained cyclic peptide inhibitors of human renin. Three goals were defined: enhanced inhibitory potency, high specificity for renin, and increased metabolic stability. Three cyclic compounds were synthesized with ring sizes 10, 12, and 14, based upon a linear hexapeptide inhibitor with a reduced amide replacing the scissile bond at the active site. When tested, the 14-membered-ring compound was as potent an inhibitor of human renin as the parent while the 12-membered-ring compound was 6-fold more potent than the parent against mouse renin. However, the 10-membered-ring compound was inactive against both renins. The lack of potency of the 10-membered compound was explained by using NMR and molecular modeling techniques. It forms another conformation in solution that is inconsistent with binding at the active site. The cyclic compounds did not inhibit either pepsin or cathepsin D significantly. The cyclic modification rendered these inhibitors significantly resistant to cleavage by chymotrypsin and thus prevented loss of activity by this enzyme. Thus, the goals of enhanced inhibitory potency, high specificity, and metabolic stability were achieved in the series of compounds.

The conformation of the cyclic portion (7-23) of naturally occurring rat atrial natriuretic factor, ANF(1-28), has been examined in sodium dodecyl sulfate (SDS) micelles using high-resolution NMR techniques. Evidence is presented which shows that ANF(7-23) has several regions of definable structure in SDS micelles which were not observed in earlier studies in bulk solvents. The 1H NMR resonances of ANF(7-23) in SDS micelles were assigned using sequential assignment techniques, and the conformational properties were analyzed primarily from proton-proton distances obtained from the quantitative analysis of two-dimensional nuclear Overhauser effect spectra. Three-dimensional structures consistent with the NMR data were generated by using distance geometry and constrained minimization/dynamics. Several similar but not identical structures were found which adequately satisfied the NMR constraints. Although none of the structures adopted a standard secondary structure, the conformations of three different sections of the peptide, 8-13, 14-17, and 18-21, were nearly identical in all of the predicted structures when individually superimposed.

The conformational and dynamic properties of a cyclic peptide designed to inhibit human renin have been examined by using NMR and molecular modeling. From a quantitative analysis of a series of two-dimensional NOE data sets, proton-proton distances were calculated. Several different methods were explored and compared to incorporate these distance constraints as well as those derived from vicinal spin-spin coupling constants into computer-generated three-dimensional structures. These methods included interactive manual manipulation of the structures to fit the NMR-determined distance constraints, distance geometry, constrained energy minimizations, and constrained molecular dynamics. The advantages and disadvantages of the methods are discussed. In addition, to gain insight into the conformations accessible to the cyclic peptide and the relative flexibility of the different parts of the molecule, molecular dynamics calculations were performed at three different temperatures. Average interproton distances and dihedral angles were obtained from the structures generated in the dynamics trajectories and compared to those obtained from the NMR experiments. Despite the four methylene groups and ether linkage contained in the cyclic portion of the peptide, our NMR results indicated a preferred conformation for the macrocyclic ring of the peptide and supported the presence of a cis Phe-Ala peptide bond. In contrast, both the molecular dynamics and NMR data indicated a considerable amount of flexibility for the remaining noncyclic portion of the molecule. These results are used to propose an explanation for the cyclic peptide's inability to inhibit human renin.

The utility of three-dimensional heteronuclear NMR spectroscopy for the assignment of 1H and 15N resonances of the inflammatory protein C5a (MW 8500), uniformly labeled with 15N, is demonstrated at a protein concentration of 0.7 mM. It is shown that dramatic simplification of the 2D nuclear Overhauser effect spectrum (NOESY) is obtained by editing with respect to the frequency of the 15N heteronucleus in a third dimension. The improved resolution in the 3D experiment largely facilitates the assignment of protein NMR spectra and allows for the determination of distance constraints from otherwise overlapping NOE cross peaks for purposes of 3D structure determination. The results show that 15N heteronuclear 3D NMR can facilitate the structure determination of small proteins and promises to be a useful tool for the study of larger systems that cannot be studied by conventional 2D NMR techniques.

Amphetamine is a strong competitive antagonist of brain synaptosomal [3H]norepinephrine ([3H]NE) uptake. Its alpha-ethyl analogue is much less active, while 2-aminotetralin and 1,2-dihydro-2-aminonaphthalene, in which the alpha-ethyl group is tied to the aromatic ring, possess about the same inhibitory potency as amphetamine. The conformational properties of these compounds in solution were studied by 1H and 13C NMR methods. Only small differences between amphetamine and alpha-ethylphenethylamine hydrochlorides were observed in the relative rotamer populations due to rotation around the C alpha -C beta bond of the side chain. In D2O the gauche conformation is slightly favored, while in CDCl3 the trans conformation is the predominant one. Conformational analysis of the alpha-ethyl group in alpha-ethylphenethylamine showed that this group exists in two equally populated conformations in both solvents. It is suggested that these conformations hinder the approach of alpha-ethylphenethylamine to the brain synaptosomal NE uptake sites.

Theoretical conformational energy calculations were carried out for the (+) and (-) isomers of the hallucinogen 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane (DOM, STP). Energies were also calculated for two analogues of DOM, 1-amino-1-(2,5-dimethoxy-4-methylbenzyl)cyclopropane and 1-(2,5-dimethoxy-4-methylphenyl)-2-methyl-2-aminopropane. This method utilized classical, empirical potential-energy functions. A previously proposed active conformational region was studied. Compounds could be ranked in order of potency based on relative conformational energies in this region. Measurement of 13C spin--lattice relaxation times (T1) for the two alpha, alpha-disubstituted DOM analogues confirmed theoretical predictions of very restricted conformational freedom for the dimethyl compound but more flexibility for the cyclopropane analogue.

A general approach is illustrated for providing detailed structural information on large enzyme/inhibitor complexes using NMR spectroscopy. The method involves the use of isotopically labeled ligands to simplify two-dimensional NOE spectra of large molecular complexes by isotope-editing techniques. With this approach, the backbone and side-chain conformations (at the P2 and P3 sites) of a tightly bound inhibitor of porcine pepsin have been determined. In addition, structural information on the active site of pepsin has been obtained. Due to the sequence homology between porcine pepsin and human renin, this structural information may prove useful for modeling renin/inhibitor complexes with the ultimate goal of designing more effective renin inhibitors. Moreover, this general approach can be applied to study other biological systems of interest such as other enzyme/inhibitor complexes, ligands bound to soluble receptors, and enzyme/substrate interactions.

A series of ristocetin analogues with modifications (OH, C=O, C=NOH, NCOCH3) at the C-1' amino group was synthesized and found to possess antibacterial activity against gram-positive bacteria and to bind to Ac2-Lys-D-Ala-D-Ala, a model for the antibiotic's site of action. Due to the lack of a positively charged amino group, the active analogues could not form a salt bridge, indicating that an electrostatic interaction between the positively charged 1'-amino group of ristocetin and the carboxylate anion of the peptide is not required for complex formation. The only compound that did not exhibit good antibacterial activity was epiristocetin aglycone (an analogue with the 1'amino group in the opposite configuration (S) as ristocetin). On the basis of NMR studies of epiristocetin aglycone in solution, the 1'-amino group is located in the proposed carboxylate binding pocket and may sterically block complex formation.