Cyclic Peptide Design Guided by Residual Dipolar Couplings, J-Couplings, and Intramolecular Hydrogen Bond Analysis.

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.

Utilizing on- and off-line monitoring tools to follow a kinetic resolution step during flow synthesis.

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.

Regio- and Enantioselective Synthesis of Azole Hemiaminal Esters by Lewis Base Catalyzed Dynamic Kinetic Resolution.

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.

Design and optimization of selective azaindole amide M1 positive allosteric modulators.

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.

Design and synthesis of thiophene dihydroisoquinolines as novel BACE1 inhibitors.

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.

Novel isoquinolone PDK1 inhibitors discovered through fragment-based lead discovery.

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.

Intramolecular Ring-Opening Decomposition of Aryl Azetidines.

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.

Biochemical characterization of recombinant hepatitis C virus nonstructural protein 4B: evidence for ATP/GTP hydrolysis and adenylate kinase activity.

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.

CBFbeta allosterically regulates the Runx1 Runt domain via a dynamic conformational equilibrium.

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.

Depletion of Gut-Resident CCR5+ Cells for HIV Cure Strategies.

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.

Discovery of Potent and Orally Bioavailable Macrocyclic Peptide-Peptoid Hybrid CXCR7 Modulators.

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 < 100 nM) and measurable passive permeability (Papp > 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.

Discovery of Clinical Candidate 1-{[(2S,3S,4S)-3-Ethyl-4-fluoro-5-oxopyrrolidin-2-yl]methoxy}-7-methoxyisoquinoline-6-carboxamide (PF-06650833), a Potent, Selective Inhibitor of Interleukin-1 Receptor Associated Kinase 4 (IRAK4), by Fragment-Based Drug Design.

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.

Discovery of a Selective Covalent Inhibitor of Lysophospholipase-like 1 (LYPLAL1) as a Tool to Evaluate the Role of this Serine Hydrolase in Metabolism.

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.

Using NMR for ligand discovery and optimization.

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.

ID NMR Methods in ligand-receptor interactions.

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.

The effect of relaxation on the epitope mapping by saturation transfer difference NMR.

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.

Conformational Flexibility of Deoxyoligonucleotide d(GGTATACC)(2) in Water.

AT-rich deoxyoligonucleotide provides a binding site possibly at the minor groove for some anti-tumor drugs by hydrophobic or Van der Waals interactions. In this paper, it is demonstrated by study of d (GGTATACC)(2) that the DNA-drug interaction may be dependent on the structural flexibility at the minor groove. The solution structure of d (GGTATACC)(2) in water is described by distance-restrained molecular dynamics calculation and it is suggested that d (GGTATACC)(2) in solution maintains the double helix of B-type with trans conformations of base to sugar and C2'-endo conformation for the deoxyribose ring. It is found that the end moieties GGT and ACC are relatively rigid while T(5) residue is flexible, which may account for the activity of the minor groove.

High throughput method for the indirect detection of intramolecular hydrogen bonding.

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.

The hepatoselective glucokinase activator PF-04991532 ameliorates hyperglycemia without causing hepatic steatosis in diabetic rats.

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 ß-cells and hepatocytes. Since activation of glucokinase in ß-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.

Label free fragment screening using surface plasmon resonance as a tool for fragment finding - analyzing parkin, a difficult CNS target.

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.