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[Image: see text] The ring strain present in azetidines can lead to undesired stability issues. Herein, we described a series of N-substituted azetidines which undergo an acid-mediated intramolecular ring-opening decomposition via nucleophilic attack of a pendant amide group. Studies were conducted to understand the decomposition mechanism enabling the design of stable analogues.

Cyclic peptides have long tantalized drug designers with their potential ability to combine the best attributes of antibodies and small molecules. An ideal cyclic peptide drug candidate would be able to recognize a protein surface like an antibody while achieving the oral bioavailability of a small molecule. It has been hypothesized that such cyclic peptides balance permeability and solubility using their solvent-dependent conformational flexibility. Herein we report a conformational deconvolution NMR methodology that combines residual dipolar couplings, J-couplings, and intramolecular hydrogen bond analysis along with conformational analysis using molecular dynamics simulations and density functional theory calculations for studying cyclic peptide conformations in both low-dielectric solvent (chloroform) and high-dielectric solvent (DMSO) to experimentally study the solvent-dependent conformational change hypothesis. Taken together, the combined experimental and computational approaches can illuminate conformational ensembles of cyclic peptides in solution and help identify design opportunities for better permeability.

Through fragment-based drug design focused on engaging the active site of IRAK4 and leveraging three-dimensional topology in a ligand-efficient manner, a micromolar hit identified from a screen of a Pfizer fragment library was optimized to afford IRAK4 inhibitors with nanomolar potency in cellular assays. The medicinal chemistry effort featured the judicious placement of lipophilicity, informed by co-crystal structures with IRAK4 and optimization of ADME properties to deliver clinical candidate PF-06650833 (compound 40). This compound displays a 5-unit increase in lipophilic efficiency from the fragment hit, excellent kinase selectivity, and pharmacokinetic properties suitable for oral administration.

In situ reaction monitoring tools offer the ability to track the progress of a synthetic reaction in real time to facilitate reaction optimization and provide kinetic/mechanistic insight. Herein, we report the utilization of flow NMR, flow IR, and other off-line spectroscopy tools to monitor the progress of a flow chemistry reaction. The on-line and off-line tools were selected to facilitate the stereoselective kinetic resolution of a key racemic monomer, which lacked a chromophore, making conventional reaction monitoring difficult. Copyright © 2016 John Wiley & Sons, Ltd.

The HIV reservoir forming at the earliest stages of infection is likely composed of CCR5+ cells, because these cells are the targets of transmissible virus. Restriction of the CCR5+ reservoir, particularly in the gut, may be needed for subsequent cure attempts. Strategies for killing or depleting CCR5+ cells have been described, but none have been tested in vivo in nonhuman primates, and the extent of achievable depletion from tissues is not known. In this study we investigate the efficacy of two novel cytotoxic treatments for targeting and eliminating CCR5+ cells in young rhesus macaques. The first, an immunotoxin consisting of the endogenous CCR5 ligand RANTES fused with Pseudomonas exotoxin (RANTES-PE38), killed CCR5+ lamina propria lymphocytes (LPLs) ex vivo, but had no detectable effect on CCR5+ LPLs in vivo. The second, a primatized bispecific antibody for CCR5 and CD3, depleted all CCR5+ cells from blood and the vast majority of such cells from the colonic mucosa (up to 96% of CD4+CCR5+). Absence of CCR5-expressing cells from blood endured for at least 1 week, while CCR5+ cells in colon were substantially replenished over the same time span. These data open an avenue to investigation of combined early ART treatment and CCR5+ reservoir depletion for cure of HIV-infected infants.

The chemokine receptor CXCR7 is an attractive target for a variety of diseases. While several small-molecule modulators of CXCR7 have been reported, peptidic macrocycles may provide advantages in terms of potency, selectivity, and reduced off-target activity. We produced a series of peptidic macrocycles that incorporate an N-linked peptoid functionality where the peptoid group enabled us to explore side-chain diversity well beyond that of natural amino acids. At the same time, theoretical calculations and experimental assays were used to track and reduce the polarity while closely monitoring the physicochemical properties. This strategy led to the discovery of macrocyclic peptide–peptoid hybrids with high CXCR7 binding affinities (Ki 5 × 10–6 cm/s). Moreover, bioactive peptide 25 (Ki = 9 nM) achieved oral bioavailability of 18% in rats, which was commensurate with the observed plasma clearance values upon intravenous administration.

We report a modular three-component dynamic kinetic resolution (DKR) that affords enantiomerically enriched hemiaminal esters derived from azoles and aldehydes. The novel and scalable reaction can be used to synthesize valuable substituted azoles in a regioselective manner by capping (e.g., acylation) of the equilibrating azole-aldehyde adduct. With the use of a prolinol-derived DMAP catalyst as the chiral Lewis base, the products can be obtained in high chemical yield and with high enantiomeric excess. The DKR was performed on a multikilogram scale to produce a tetrazole prodrug fragment for a leading clinical candidate that posed formidable synthesis challenges.

Lysophospholipase-like 1 (LYPLAL1) is an uncharacterized metabolic serine hydrolase. Human genome-wide association studies link variants of the gene encoding this enzyme to fat distribution, waist-to-hip ratio, and nonalcoholic fatty liver disease. We describe the discovery of potent and selective covalent small-molecule inhibitors of LYPLAL1 and their use to investigate its role in hepatic metabolism. In hepatocytes, selective inhibition of LYPLAL1 increased glucose production supporting the inference that LYPLAL1 is a significant actor in hepatic metabolism. The results provide an example of how a selective chemical tool can contribute to evaluating a hypothetical target for therapeutic intervention, even in the absence of complete biochemical characterization.

In situ reaction monitoring tools offer the ability to track the progress of a synthetic reaction in real time to facilitate reaction optimization and provide kinetic/mechanistic insight. Herein, we report the utilization of flow NMR, flow IR, and other off-line spectroscopy tools to monitor the progress of a flow chemistry reaction. The on-line and off-line tools were selected to facilitate the stereoselective kinetic resolution of a key racemic monomer, which lacked a chromophore, making conventional reaction monitoring difficult. Copyright © 2016 John WileySons, Ltd.

We report a modular three-component dynamic kinetic resolution (DKR) that affords enantiomerically enriched hemiaminal esters derived from azoles and aldehydes. The novel and scalable reaction can be used to synthesize valuable substituted azoles in a regioselective manner by capping (e.g., acylation) of the equilibrating azole-aldehyde adduct. With the use of a prolinol-derived DMAP catalyst as the chiral Lewis base, the products can be obtained in high chemical yield and with high enantiomeric excess. The DKR was performed on a multikilogram scale to produce a tetrazole prodrug fragment for a leading clinical candidate that posed formidable synthesis challenges.

The heterodimer HIF-1α (hypoxia inducible factor)/HIF-β (also known as ARNT-aryl hydrocarbon nuclear translocator) is a key mediator of cellular response to hypoxia. The interaction between these monomer units can be modified by the action of small molecules in the binding interface between their C-terminal heterodimerization (PasB) domains. Taking advantage of the presence of several cysteine residues located in the allosteric cavity of HIF-1α PasB domain, we applied a cysteine-based reactomics "hotspot identification" strategy to locate regions of HIF-1α PasB domain critical for its interaction with ARNT. COMPOUND 5 was identified using a mass spectrometry-based primary screening strategy and was shown to react specifically with Cys255 of the HIF-1α PasB domain. Biophysical characterization of the interaction between PasB domains of HIF-1α and ARNT revealed that covalent binding of COMPOUND 5 to Cys255 reduced binding affinity between HIF-1α and ARNT PasB domains approximately 10-fold. Detailed NMR structural analysis of HIF-1α-PasB-COMPOUND 5 conjugate showed significant local conformation changes in the HIF-1α associated with key residues involved in the HIF-1α/ARNT PasB domain interaction as revealed by the crystal structure of the HIF-1α/ARNT PasB heterodimer. Our screening strategy could be applied to other targets to identify pockets surrounding reactive cysteines suitable for development of small molecule modulators of protein function.

The Per-Arnt-Sim (PAS) domains of hypoxia-inducible transcription factors (HIF) mediate heterodimer formation between the HIF-α forms that are induced in the event of cellular hypoxia and the constitutive HIF-β variants. Previous efforts towards structural characterization of the HIF-1α PAS domains were limited by protein stability. Using homology modeling based on the published crystal structure of the PAS-B domain of the homologous protein HIF-2α in complex with the partner HIF-β (also known as ARNT), we have identified a variant of HIF-1α with improved solubility, monodispersity and stability. Purified solutions of the PAS-B domains of HIF-1α and HIF-2α differ in their propensity for homodimer formation. In an attempt to understand the structural basis for this difference, and to document the structural changes that accompany homodimer formation, we have undertaken a comparative NMR study of the PAS-B domains of HIF-1α and HIF-2α and mutants of HIF-1α that mimic the behavior of HIF-2α. The NMR spectra of all of these domains are very similar, consistent with the similarity of their amino acid sequences. However, the greater propensity of the HIF-1α PAS-B domain to form dimers as the concentration was increased allowed us to determine the site of homodimerization and pointed towards possible sequence changes in HIF-1α that might discourage the formation of homodimers.

HIV-1 reverse transcriptase (RT) RNase H (HIV-RH) is a key target of anti-AIDS drugs. Metal-chelating compounds are an important class of chemicals in pharmacological drug discovery, especially in relation to HIV-RT and the highly-related HIV-integrase. The correlation between the metal-chelating properties and enzyme activities of the metal chelators is always of high scientific interest, as an understanding of this may accelerate the rational optimization of this class of inhibitors. Our NMR data show that Mg2+ and Ca2+ bind specifically to the active site of the RNase H domain and two Mg2+ ions sequentially bind one molecule of RNase H. We also demonstrate here, using saturated and unsaturated tricyclic N-hydroxypyridones designed to block the active site, that the primary binding sites and affinities of divalent metal ions are correlated with the structures of the chelating motifs. Chemical shift perturbation studies of protein/metal-ion/compound ternary complexes also indicate that divalent metal ions play important roles for the specific interaction of the compounds with the RNase H active site.

Selective activation of the M1 receptor via a positive allosteric modulator (PAM) is a new approach for the treatment of the cognitive impairments associated with schizophrenia and Alzheimer's disease. A novel series of azaindole amides and their key pharmacophore elements are described. The nitrogen of the azaindole core is a key design element as it forms an intramolecular hydrogen bond with the amide N-H thus reinforcing the bioactive conformation predicted by published SAR and our homology model. Representative compound 25 is a potent and selective M1 PAM that has well aligned physicochemical properties, adequate brain penetration and pharmacokinetic (PK) properties, and is active in vivo. These favorable properties indicate that this series possesses suitable qualities for further development and studies.

Core binding factors (CBFs) are heterodimeric transcription factors consisting of a DNA-binding CBFalpha subunit and non-DNA-binding CBFbeta subunit. The CBFbeta subunit increases the affinity of the DNA-binding Runt domain of CBFalpha for DNA while making no direct contacts to the DNA. We present evidence for conformational exchange in the S-switch region in a Runt domain-DNA complex that is quenched upon CBFbeta binding. Analysis of (15)N backbone relaxation parameters shows that binding of CBFbeta reduces the backbone dynamics in the microsecond-to-millisecond time frame for several regions of the Runt domain that make energetically important contacts with the DNA. The DNA also undergoes conformational exchange in the Runt domain-DNA complex that is quenched in the presence of CBFbeta. Our results indicate that allosteric regulation by the CBFbeta subunit is mediated by a shift in an existing dynamic conformational equilibrium of both the Runt domain and DNA.

Residual dipolar couplings (RDCs), in combination with molecular order matrix calculations, were used to unambiguously determine the complete relative stereochemistry of an organic compound with five stereocenters. Three simple one-dimensional experiments were utilized for the measurements of (13)C-(1)H, (13)C-(19)F, (19)F-(1)H, and (1)H-(1)H RDCs. The order matrix calculation was performed on each chiral isomer independently. The fits were evaluated by the comparison of the root-mean-square deviation (rmsd) of calculated and measured RDCs. The order tensor simulations based on two different sets of RDC data collected with phage and bicelles are consistent. The resulting stereochemical assignments of the stereocenters obtained from using only RDCs are in perfect agreement with those obtained from the single-crystal X-ray structure. Six RDCs are found to be necessary to run the simulation, and seven are the minimum to get an acceptable result for the investigated compound. It was also shown that (13)C-(1)H and (1)H-(1)H RDCs, which are the easiest to measure, are also the most important and information-rich data for the order matrix calculation. The effect of each RDC on the calculation depends on the location of the corresponding vector in the structure. The direct RDC of a stereocenter is important to the configuration determination, but the configuration of stereocenters devoid of protons can also be obtained from analysis of nearby RDCs.

The effect of longitudinal relaxation of ligand protons on saturation transfer difference (STD) was investigated by using a known binding system, dihydrofolate reductase and trimethoprim. The results indicate that T1 relaxation of ligand protons has a severe interference on the epitope map derived from a STD measurement. When the T1s of individual ligand protons are distinctly different, STD experiments may not give an accurate epitope map for the ligand–target interactions. Measuring the relaxation times prior to mapping is strongly advised. A saturation time shorter than T1s is suggested for improving the potential epitope map. Reduction in temperature was seen to enhance the saturation efficiency in small to medium size targets.

The drug discovery process often involves the screening of compound libraries to identify drug candidates capable of binding to target macromolecules. New approaches in biological and chemical research are driving a change in the pharmaceutical industry. Recent advances in NMR spectroscopy such as affinity NMR techniques, which detect binding of a small molecule with a “receptor”, have been shown to be valuable tools to perform rapid screening of compounds for biological activity. These NMR observable events include using relaxation, chemical shift perturbations, translational diffusion, and magnetization transfer. These one dimensional NMR methods increase both the throughput of screening and yield crucial data on the mode of binding. The practical utility of these techniques will be described.

A suite of multiple quantum (MQ) HCN-based pulse sequences has been developed for the purpose of collecting dipolar coupling data in labeled nucleic acids. All the pulse sequences are based on the robust MQ-HCN experiment which has been utilized for assignment purposes in labeled nucleic acids for a number of years and provides much-needed resolution for the dipolar coupling measurements. We have attempted to collect multiple couplings centered on the 13C1′ and 13C6/8 positions. Six pulse sequences are described, one each for measurement of one-bond 13C1′-1H1′ and 13C6/8-1H6/8 couplings, one for measurement of one-bond 13C1′-15N and two-bond 1H1′-15N couplings, one for measurement of one-bond13C6/8-15N and two-bond 1H6/8-15N couplings, one for measurement of one-bond 13C1′-13C2′ and two-bond 1H1′-13C2′ couplings, and one for measurement of one-bond 13C6-13C5 and two-bond 1H6-13C5 couplings in the bases of C and T. These sequences are demonstrated for a labeled 18 bp DNA duplex in a 47 kDa ternary complex of DNA, CBFβ, and the CBFα Runt domain, thus clearly demonstrating the robustness of the pulse sequences even for a very large complex.

A supercritical fluid chromatography method was developed for the detection of intramolecular hydrogen bonds in pharmaceutically relevant molecules. The identification of compounds likely to form intramolecular hydrogen bonds is an important drug design consideration given the correlation of intramolecular hydrogen bonding with increased membrane permeability. The technique described here correlates chromatographic retention with the exposed polarity of a molecule. Molecules that can form an intramolecular hydrogen bond can hide their polarity and therefore exhibit lower retention than similar compounds that cannot. By use of a pairwise analysis strategy, intramolecular hydrogen bonds are identified within a test set of compounds with diverse topologies. The chromatographic results are confirmed by NMR chemical shift and temperature coefficient studies.

Hyperglycemia resulting from type 2 diabetes mellitus (T2DM) is the main cause of diabetic complications such as retinopathy and neuropathy. A reduction in hyperglycemia has been shown to prevent these associated complications supporting the importance of glucose control. Glucokinase converts glucose to glucose-6-phosphate and determines glucose flux into the β-cells and hepatocytes. Since activation of glucokinase in β-cells is associated with increased risk of hypoglycemia, we hypothesized that selectively activating hepatic glucokinase would reduce fasting and postprandial glucose with minimal risk of hypoglycemia. Previous studies have shown that hepatic glucokinase overexpression is able to restore glucose homeostasis in diabetic models; however, these overexpression experiments have also revealed that excessive increases in hepatic glucokinase activity may also cause hepatosteatosis. Herein we sought to evaluate whether liver specific pharmacological activation of hepatic glucokinase is an effective strategy to reduce hyperglycemia without causing adverse hepatic lipids changes. To test this hypothesis, we evaluated a hepatoselective glucokinase activator, PF-04991532, in Goto-Kakizaki rats. In these studies, PF-04991532 reduced plasma glucose concentrations independent of changes in insulin concentrations in a dose-dependent manner both acutely and after 28 days of sub-chronic treatment. During a hyperglycemic clamp in Goto-Kakizaki rats, the glucose infusion rate was increased approximately 5-fold with PF-04991532. This increase in glucose infusion can be partially attributed to the 60% reduction in endogenous glucose production. While PF-04991532 induced dose-dependent increases in plasma triglyceride concentrations it had no effect on hepatic triglyceride concentrations in Goto-Kakizaki rats. Interestingly, PF-04991532 decreased intracellular AMP concentrations and increased hepatic futile cycling. These data suggest that hepatoselective glucokinase activation may offer glycemic control without inducing hepatic steatosis supporting the evaluation of tissue specific activators in clinical trials.

Core binding factors (CBFs) play key roles in several developmental pathways and in human disease. CBFs consist of a DNA binding CBFalpha subunit and a non-DNA binding CBFbeta subunit that increases the affinity of CBFalpha for DNA. We performed sedimentation equilibrium analyses to unequivocally establish the stoichiometry of the CBFalpha:beta:DNA complex. Dissociation constants for all four equilibria involving the CBFalpha Runt domain, CBFbeta, and DNA were defined. Conformational changes associated with interactions between CBFalpha, CBFbeta, and DNA were monitored by nuclear magnetic resonance and circular dichroism spectroscopy. The data suggest that CBFbeta 'locks in' a high affinity DNA binding conformation of the CBFalpha Runt domain.

Utilizing a structure based design approach, combined with extensive medicinal chemistry execution, highly selective, potent and novel BACE1 inhibitor 8 (BACE1 Alpha assay IC50=8nM) was made from a weak μM potency hit in an extremely efficient way. The detailed SAR and general design approaches will be discussed.

Phosphoinositide-dependent kinase-1 (PDK1) is a critical enzyme in the PI3K/AKT pathway and to the activation of AGC family protein kinases, including S6K, SGK, and PKC. Dysregulation of this pathway plays a key role in cancer cell growth, survival and tumor angiogenesis. As such, inhibitors of PDK1 offer the promise of a new therapeutic modality for cancer treatment. Fragment based drug screening has recently become a viable entry point for hit identification. In this work, NMR spectroscopy fragment screening of PDK1 afforded novel chemotypes as orthogonal starting points from HTS screening hits. Compounds identified as hits by NMR spectroscopy were tested in a biochemical assay, and fragments with activity in both assays were clustered. The Pfizer compound file was mined via substructure and 2D similarity search, and the chemotypes were prioritized by ligand efficiency (LE), SAR mining, chemical attractiveness, and chemical enablement of promising vectors. From this effort, an isoquinolone fragment hit, 5 (IC(50) 870 μM, LE = 0.39), was identified as a novel, ligand efficient inhibitor of PDK1 and a suitable scaffold for further optimization. Initially in the absence of crystallographic data, a fragment growing approach efficiently explored four vectors of the isoquinolone scaffold via parallel synthesis to afford a compound with crystallographic data, 16 (IC(50) 41.4 μM, LE = 0.33). Subsequent lead optimization efforts provided 24 (IC(50) 1.8 μM, LE = 0.42), with greater than fivefold selectivity against other key pathway kinases.

While nonstructural protein 4B (NS4B) from hepatitis C virus (HCV) is absolutely required for viral propagation, a full understanding of the enzymatic properties of this protein is lacking. Previous studies suggest that NS4B is located at the endoplasmic reticulum and that the protein structure consists of four central transmembrane domains with the N- and C-termini located in the cytoplasm of the host cell. To characterize the enzymatic activity of NS4B, the full-length protein with a C-terminal His tag was expressed in Sf9 insect cells and stabilized with nonionic detergents during purification. Chemical cross-linking experiments using GTP-gamma-azidoanilide and ATP-gamma-azidoanilide and equilibrium binding analyses with GTPgammaS and ATPgammaS show that both GTP and ATP are bound by NS4B, with ATP displaying a higher affinity. Analyses of enzymatic reactions catalyzed by NS4B indicate that the terminal phosphate groups of ATP, GTP, and GDP are removed to produce ADP, GDP, and GMP, respectively. The k(cat) for hydrolysis of GTP by purified NS4B compared favorably with the k(cat) for hydrolysis of GTP by Ras-p21 in the absence of GTPase activating proteins (GAPs). In addition to the hydrolysis of NTP and NDP substrates, adenylate kinase activity was detected in purified preparations of NS4B with the reverse reaction 2ADP --ATP + ADP, yielding a larger k(cat) compared to that of the forward reaction ATP + AMP --2ADP. These studies suggest that HCV NS4B possesses both adenylate kinase activity and nucleotide hydrolase activity. Mutation of amino acids in the Walker A and B motifs of NS4B resulted in decreased affinity for both GTPgammaS and ATPgammaS as well as decreased ATP hydrolysis and AK activity.

Residual dipolar couplings (RDCs) induced by anisotropic media are a powerful tool for the structure determination of biomolecules through NMR spectroscopy. Recent advances have proven it to be a valuable tool for determination of the stereochemistry of organic molecules. By simple inspection or order matrix calculations, RDCs provide unambiguous information about the relative configurations or complete stereochemistry of organic compounds.

A novel methodology using the order matrix calculation to determine the absolute sign of spin–spin couplings based on the structure of organic compounds is presented. The sign of the residual dipolar coupling (RDC) depends on the sign of corresponding scalar spin–spin coupling constant and the sign of the RDC has a dramatic influence on the order matrix calculation. Therefore, the sign of the spin–spin coupling constant can be obtained by an order matrix calculation through the corresponding RDC. Six types of spin–spin coupling constants, including 2J(H,H), 1J(C,F), 2J(C,F), 3J(C,F), 2J(F,H) and 3J(F,H), were obtained simultaneously. Except for 3J(C,F) where the measured RDCs have very small magnitudes, the signs were determined unambiguously. Copyright © 2004 John Wiley & Sons, Ltd.

Several recent technology-driven advances in the area of NMR have rekindled an interest in the application of the technology to problems in drug discovery and development. A unique aspect of NMR is that it has applicability in broadly different areas of the drug discovery and optimization processes. NMR techniques for screening aimed at the discovery of novel ligands or low molecular weight structures for fragment-based build up procedures are being applied commonly in the industry. Application of NMR in structure-guided drug design and metabonomics are also becoming routine. We present an overview of some of the most recent NMR developments in these areas.

The crucial step in drug discovery is the identification of a lead compound from a vast chemical library by any number of screening techniques. NMR-based screening has the advantage of directly detecting binding of a compound to the target. The spectra resulting from these screens can also be very complex and difficult to analyze, making this an inefficient process. We present here a method, RAMPED-UP NMR, (Rapid Analysis and Multiplexing of Experimentally Discriminated Uniquely Labeled Proteins using NMR) which generates simple spectra which are easy to interpret and allows several proteins to be screened simultaneously. In this method, the proteins to be screened are uniquely labeled with one amino acid type. There are several benefits derived from this unique labeling strategy: the spectra are greatly simplified, resonances that are most likely to be affected by binding are the only ones observed, and peaks that yield little or no information upon binding are eliminated, allowing the analysis of multiple proteins easily and simultaneously. We demonstrate the ability of three different proteins to be analyzed simultaneously for binding to two different ligands. This method will have significant impact in the use of NMR spectroscopy for both the lead generation and lead optimization phases of drug discovery by its ability to increase screening throughput and the ability to examine selectivity. To the best of our knowledge, this is the first time in any format that multiple proteins can be screened in one tube.

Core-binding factors (CBFs) are a small family of heterodimeric transcription factors that play critical roles in several developmental pathways and in human disease. Mutations in CBF genes are found in leukemias, bone disorders, and gastric cancers. CBFs consist of a DNA-binding CBF alpha subunit (Runx1, Runx2, or Runx3) and a non-DNA-binding CBF beta subunit. CBF alpha binds DNA in a sequence-specific manner, whereas CBF beta enhances DNA binding by CBF alpha. Both DNA binding and heterodimerization with CBF beta are mediated by a single domain in the CBF alpha subunits known as the "Runt domain." We analyzed the energetic contribution of amino acids in the Runx1 Runt domain to heterodimerization with CBF beta. We identified two energetic "hot spots" that were also found in a similar analysis of CBF beta (Tang, Y.-Y., Shi, J., Zhang, L., Davis, A., Bravo, J., Warren, A. J., Speck, N. A., and Bushweller, J. H. (2000) J. Biol. Chem. 275, 39579-39588). The importance of the hot spot residues for Runx1 function was demonstrated in in vivo transient transfection assays. These data refine the structural analyses and further our understanding of the Runx1-CBF beta interface.

Core-binding factors (CBFs) are a small family of heterodimeric transcription factors that play critical roles in hematopoiesis and in the development of bone, stomach epithelium, and proprioceptive neurons. Mutations in CBF genes are found in leukemias, bone disorders, and gastric cancer. CBFs consist of a DNA-binding CBF alpha subunit and a non-DNA-binding CBF beta subunit. DNA binding and heterodimerization with CBF beta are mediated by the Runt domain in CBF alpha. Here we report an alanine-scanning mutagenesis study of the Runt domain that targeted amino acids identified by structural studies to reside at the DNA or CBF beta interface, as well as amino acids mutated in human disease. We determined the energy contributed by each of the DNA-contacting residues in the Runt domain to DNA binding both in the absence and presence of CBF beta. We propose mechanisms by which mutations in the Runt domain found in hematopoietic and bone disorders affect its affinity for DNA.

A novel method for the determination of the relative stereochemistry of six-membered chairlike ring molecules by residual dipolar couplings is presented. C−H residual dipolar couplings were used to investigate the relative stereochemistry of 4,6-O-ethylidene-d-glucopyranose. For this and similar systems it is not necessary to acquire redundant dipolar couplings and to calculate the orientation order tensor. The presented methodology is a paradigmatic leap for the determination of the relative stereochemistry or remote stereochemistry in this kind of fused ring system. Residual dipolar coupling data were collected by 1D and 2D direct-measurement heteronuclear multiple quantum coherence (HMQC) spectroscopy. It was demonstrated that direct measurement of HMQC was quick and accurate for small molecules at natural abundance.

The drug discovery process relies on characterizing structure-activity relationships, since specific ligand-target interactions often result in important biological functions. Measuring diffusion coefficients by nuclear magnetic resonance spectroscopy is a useful way to study binding, because changes can be detected when a small ligand interacts with a macromolecular target. Diffusion coefficients can be miscalculated, however, due to magnetization transfer between the receptor and ligand. This transferred nuclear Overhauser effect (trNOE) disrupts the observed signal decay due to diffusion as a function of the experimental diffusion time. Since longer diffusion times also selectively edit free ligand signal, the measured diffusion coefficients become biased toward the fraction of bound ligand. Despite this discrepancy, under these experimental conditions, the trNOE selectively influences the measured signals of binding ligands and can be used to gain insight into ligand-protein interactions. These phenomena have been studied for caffeine and L-tryptophan, which bind to human serum albumin, and the antimalarial agent trimethoprim, which interacts with dihydrofolate reductase. The results provide insight into the nature of ligand-protein binding and are thus useful for elucidating the molecular features of the ligand that interact with the protein.

The drug discovery process often involves the screening of compound libraries to identify drug candidates capable of binding to target macromolecules. New approaches in biological and chemical research are driving a change in the pharmaceutical industry. Recent advances in NMR spectroscopy such as affinity NMR techniques, which detect binding of a small molecule with a "receptor", have been shown to be valuable tools to perform rapid screening of compounds for biological activity. These NMR observable events include using relaxation, chemical shift perturbations, translational diffusion, and magnetization transfer. These one dimensional NMR methods increase both the throughput of screening and yield crucial data on the mode of binding. The practical utility of these techniques will be described.

A novel method based on diffusion NMR for the epitope mapping of ligand binding is presented. The intermolecular NOE builds up during a long diffusion period and creates a deviation from the linearity. The ligand proton nearest the protein generates the strongest NOE from protein during the diffusion period and has the largest deviation. Therefore, this diffusion artifact can be used to characterize the ligand binding epitope. The concept was investigated using dihydrofolate reductase (DHFR) and its ligand trimethoprim (TMP), and the epitope map of TMP on DHFR generated with this method is in excellent agreement with the structural and dynamic studies by crystallography and NMR, as well as the medicinal chemistry results.

A suite of multiple quantum (MQ) HCN-based pulse sequences has been developed for the purpose of collecting dipolar coupling data in labeled nucleic acids. All the pulse sequences are based on the robust MQ-HCN experiment which has been utilized for assignment purposes in labeled nucleic acids for a number of years and provides much-needed resolution for the dipolar coupling measurements. We have attempted to collect multiple couplings centered on the 13C1' and 13C6/8 positions. Six pulse sequences are described, one each for measurement of one-bond 13C1'-1H1' and 13C6/8-1H6/8 couplings, one for measurement of one-bond 13C1'-15N and two-bond 1H1'-15N couplings, one for measurement of one-bond 13C6/8-15N and two-bond 1H6/8-15N couplings, one for measurement of one-bond 13C1'- 13C2' and two-bond 1H1'-13C2' couplings, and one for measurement of one-bond 13C6-13C5 and two-bond 1H6-13C5 couplings in the bases of C and T. These sequences are demonstrated for a labeled 18 bp DNA duplex in a 47 kDa ternary complex of DNA, CBFbeta, and the CBFalpha Runt domain, thus clearly demonstrating the robustness of the pulse sequences even for a very large complex.

Surface Plasmon Resonance (SPR) is rarely used as a primary High-throughput Screening (HTS) tool in fragment-based approaches. With SPR instruments becoming increasingly high-throughput it is now possible to use SPR as a primary tool for fragment finding. SPR becomes, therefore, a valuable tool in the screening of difficult targets such as the ubiquitin E3 ligase Parkin. As a prerequisite for the screen, a large number of SPR tests were performed to characterize and validate the active form of Parkin. A set of compounds was designed and used to define optimal SPR assay conditions for this fragment screen. Using these conditions, more than 5000 pre-selected fragments from our in-house library were screened for binding to Parkin. Additionally, all fragments were simultaneously screened for binding to two off target proteins to exclude promiscuous binding compounds. A low hit rate was observed that is in line with hit rates usually obtained by other HTS screening assays. All hits were further tested in dose responses on the target protein by SPR for confirmation before channeling the hits into Nuclear Magnetic Resonance (NMR) and other hit-confirmation assays.