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Diabetes produces a chronic inflammatory state that contributes to the development of vascular disease and impaired wound healing. Despite the known individual and societal impacts of diabetic ulcers, there are limited therapies effective at improving healing. Stromal cell-derived factor 1α (SDF-1α) is a CXC chemokine that functions via activation of the CXC chemokine receptor type 4 (CXCR4) receptor to recruit hematopoietic cells to locations of tissue injury and promote tissue repair. The expression of SDF-1α is reduced in diabetic wounds, suggesting a potential contribution to wound healing impairment and presenting the CXCR4 receptor as a target for therapeutic investigations. We developed a high-throughput β-arrestin recruitment assay and conducted structure–activity relationship (SAR) studies to screen compounds for utility as CXCR4 agonists. We identified CXCR4 agonist UCUF-728 from our studies and further validated its activity in vitro in diabetic fibroblasts. UCUF-728 reduced overexpression of miRNA-15b and miRNA-29a, negative regulators of angiogenesis and type I collagen production, respectively, in diabetic fibroblasts. In vivo, UCUF-728 reduced the wound closure time by 36% and increased the evidence of angiogenesis in diabetic mice. Together, this work demonstrates the clinical potential of small molecule CXCR4 agonists as novel therapies for pathologic wound healing in diabetes.

Non-alcoholic fatty liver disease (NAFLD) has a large impact on global health. At the onset of disease, NAFLD is characterized by hepatic steatosis defined by the accumulation of triglycerides stored as lipid droplets. Developing therapeutics against NAFLD and progression to non-alcoholic steatohepatitis (NASH) remains a high priority in the medical and scientific community. Drug discovery programs to identify potential therapeutic compounds have supported high throughput/high-content screening of in vitro human-relevant models of NAFLD to accelerate development of efficacious anti-steatotic medicines. Human induced pluripotent stem cell (hiPSC) technology is a powerful platform for disease modeling and therapeutic assessment for cell-based therapy and personalized medicine. In this study, we applied AstraZeneca&rsquo, s chemogenomic library, hiPSC technology and multiplexed high content screening to identify compounds that significantly reduced intracellular neutral lipid content. Among 13,000 compounds screened, we identified hits that protect against hiPSC-derived hepatic endoplasmic reticulum stress-induced steatosis by a mechanism of action including inhibition of the cyclin D3-cyclin-dependent kinase 2-4 (CDK2-4)/CCAAT-enhancer-binding proteins (C/EBP&alpha, )/diacylglycerol acyltransferase 2 (DGAT2) pathway, followed by alteration of the expression of downstream genes related to NAFLD. These findings demonstrate that our phenotypic platform provides a reliable approach in drug discovery, to identify novel drugs for treatment of fatty liver disease as well as to elucidate their underlying mechanisms.

The chemokine system plays an important role in mediating a proinflammatory microenvironment for tumor growth in hepatocellular carcinoma (HCC). The CXCR6 receptor and its natural ligand CXCL16 are expressed at high levels in HCC cell lines and tumor tissues and receptor expression correlates with increased neutrophils in these tissues contributing to poor prognosis in patients. Availability of pharmacologcal tools targeting the CXCR6/CXCL16 axis are needed to elucidate the mechanism whereby neutrophils are affected in the tumor environment. We report the discovery of a series of small molecules with an exo-[3.3.1]azabicyclononane core. Our lead compound 81 is a potent (EC50 = 40 nM) and selective orally bioavailable small molecule antagonist of human CXCR6 receptor signaling that significantly decreases tumor growth in a 30-day mouse xenograft model of HCC.

The structure–activity relationships of 2-(piperidin-3-yl)-1H-benzimidazoles, 2-morpholine and 2-thiomorpholin-2-yl-1H-benzimidazoles are described. In the lead optimization process, the pKa and/or log P of benzimidazole analogs were reduced either by attachment of polar substituents to the piperidine nitrogen or incorporation of heteroatoms into the piperidine heterocycle. Compounds 9a and 9b in the morpholine series and 10g in the thiomorpholine series demonstrated improved selectivity and CNS profiles compared to lead compound 2 and these are potential candidates for evaluation as sedative hypnotics.

Structure–activity relationship studies were conducted to reduce CYP2D6-mediated metabolism in a series of indene H1-antihistamines. Reductions in pKa via incorporation of a β-fluoro substituent or a heteroaryl moiety were shown to reduce contributions to metabolism through this pathway. Several compounds, including 8l, 8o, and 12f were identified with promising primary in vitro profiles and reduced biotransformation via CYP2D6.

In this report, the strategy and outcome of expanding SAR exploration to improve solubility and metabolic stability are discussed. Compound 35 exhibited excellent potency, selectivity over A(1) and improved solubility of >4 mg/mL at pH 8.0. In addition, compound 35 had good metabolic stability with a scaled intrinsic clearance of 3 mL/min/kg (HLM) and demonstrated efficacy in the haloperidol induced catalepsy model.

A series of N-pyrimidinyl-2-phenoxyacetamide adenosine A(2A) antagonists is described. SAR studies led to compound 14 with excellent potency (K(i) = 0.4 nM), selectivity (A(1)/A(2A) > 100), and efficacy (MED 10 mg/kg p.o.) in the rat haloperidol-induced catalepsy model for Parkinson's disease.

alpha-Neurotoxins bind with high affinity to alpha-gamma and alpha-delta subunit interfaces of the nicotinic acetylcholine receptor. Since this high affinity complex likely involves a van der Waals surface area of approximately 1200 A(2) and 25-35 residues on the receptor surface, analysis of side chains should delineate major interactions and the orientation of bound alpha-neurotoxin. Three distinct regions on the gamma subunit, defined by Trp(55), Leu(119), Asp(174), and Glu(176), contribute to alpha-toxin affinity. Of six charge reversal mutations on the three loops of Naja mossambica mossambica alpha-toxin, Lys(27) --> Glu, Arg(33) --> Glu, and Arg(36) --> Glu in loop II reduce binding energy substantially, while mutations in loops I and III have little effect. Paired residues were analyzed by thermodynamic mutant cycles to delineate electrostatic linkages between the six alpha-toxin charge reversal mutations and three key residues on the gamma subunit. Large coupling energies were found between Arg(33) at the tip of loop II and gammaLeu(119) (-5.7 kcal/mol) and between Lys(27) and gammaGlu(176) (-5.9 kcal/mol). gammaTrp(55) couples strongly to both Arg(33) and Lys(27), whereas gammaAsp(174) couples minimally to charged alpha-toxin residues. Arg(36), despite strong energetic contributions, does not partner with any gamma subunit residues, perhaps indicating its proximity to the alpha subunit. By analyzing cationic, neutral and anionic residues in the mutant cycles, interactions at gamma176 and gamma119 can be distinguished from those at gamma55.

High-throughput screening (HTS) is a powerful approach to drug discovery, but many lead compounds are found to be unsuitable for use in vivo after initial screening. Screening in small animals like C. elegans can help avoid these problems, but this system has been limited to screens with low-throughput or no specific molecular target. We report the first in vivo 1536-well plate assay for a specific genetic pathway in C. elegans. Our assay measures induction of a gene regulated by SKN-1, a master regulator of detoxification genes. SKN-1 inhibitors will be used to study and potentially reverse multidrug resistance in parasitic nematodes. Screens of two small commercial libraries and the full Molecular Libraries Small Molecule Repository (MLSMR) of ∼364,000 compounds validate our platform for ultra HTS. Our platform overcomes current limitations of many whole-animal screens and can be widely adopted for other inducible genetic pathways in nematodes and humans.

Analogs of the known H(1)-antihistamine R-dimethindene with suitable selectivity for key GPCRs, P450 enzymes and hERG channel were assessed for metabolism profile and in vivo properties. Several analogs were determined to exhibit diverse metabolism. One of these compounds, 10a, showed equivalent efficacy in a rat EEG/EMG model to a previously identified clinical candidate and a potentially superior pharmacokinetic profile as determined from a human microdose study.

A series of indene analogs of the H1-antihistamine (−)-R-dimethindene was evaluated for selectivity in the search for potentially improved sedative-hypnotics. Variation of the 6-substitutent in the indene core in combination with a pendant electron rich heterocycle led to the identification of several potent H1-antihistamines with desirable selectivity over CYP enzymes, the M1 muscarinic receptor and the hERG channel. These compounds were candidates for further ADME profiling and in vivo evaluation.

SAR of lead benzothiophene H1-antihistamine 2 was explored to identify backup candidates with suitable pharmacokinetic profiles for an insomnia program. Several potent and selective H1-antihistamines with a range of projected half-lives in humans were identified. Compound 16d had a suitable human half-life as demonstrated in a human microdose study, but variability in pharmacokinetic profile, attributed to metabolic clearance, prevented further development of this compound. Compound 28b demonstrated lower predicted clearance in preclinical studies, and may represent a more suitable backup compound.

We introduce a novel experimental method to determine both the extent of ex vivo receptor occupancy of administered compound and its dissociation rate constant (k4). [Here, we reference k4 as the rate of offset of unlabeled ligand in convention with Motulsky and Mahan (1)]. We derived a kinetic rate equation based on the dissociation rate constant for an unlabeled compound competing for the same site as a labeled compound and describe a model to simulate fractional occupancy. To validate our model, we performed in vitro kinetics and ex vivo occupancy experiments in rat cortex with varying concentrations of (R)-dimethindene, a sedating antihistamine. Brain tissue was removed at various times post oral administration, and histamine H1 receptor ligand [3H]-doxepin binding to homogenates from drug-treated or vehicle-treated rats was measured at multiple time points at room temperature. Fractional occupancy and k4 for (R)-dimethindene binding to H1 receptors were calculated by using our proposed model. Rats dosed with 30 and 60 mg/kg (R)-dimethindene showed 42% and 67% occupancy of central H1 receptors, respectively. These results were comparable to occupancy data determined by equilibrium radioligand binding. In addition, drug k4 rate determined by using our ex vivo method was equivalent to k4 determined by in vitro competition kinetics (dissociation half-life t(1/2) approximately 30 min). The outlined method can be used to assess, by simulation and experiment, occupancy for compounds based on dissociation rate constants and contributes to current efforts in drug optimization to profile antagonist efficacy in terms of its kinetic drug-target binding parameters. Data described by the method may be analyzed with commercially available software. Suggested fitting procedures are given in the appendix.

The benzimidazole core of the selective non-brain-penetrating H 1 -antihistamine mizolastine was used to identify a series of brain-penetrating H 1 -antihistamines for the potential treatment of insomnia. Using cassette PK studies, brain-penetrating H 1 -antihistamines were identified and in vivo efficacy was demonstrated in a rat EEG/EMG model. Further optimization focused on strategies to attenuate an identified hERG liability, leading to the discovery of 4i with a promising in vitro profile.

Peptide toxins selective for particular subunit interfaces of the nicotinic acetylcholine receptor have proven invaluable in assigning candidate residues located in the two binding sites and for determining probable orientations of the bound peptide. We report here on a short alpha-neurotoxin from Naja mossambica mossambica (NmmI) that, similar to other alpha-neurotoxins, binds with high affinity to alphagamma and alphadelta subunit interfaces (KD approximately 100 pM) but binds with markedly reduced affinity to the alphaepsilon interface (KD approximately 100 nM). By constructing chimeras composed of portions of the gamma and epsilon subunits and coexpressing them with wild type alpha, beta, and delta subunits in HEK 293 cells, we identify a region of the subunit sequence responsible for the difference in affinity. Within this region, gammaPro-175 and gammaGlu-176 confer high affinity, whereas Thr and Ala, found at homologous positions in epsilon, confer low affinity. To identify an interaction between gammaGlu-176 and residues in NmmI, we have examined cationic residues in the central loop of the toxin and measured binding of mutant toxin-receptor combinations. The data show strong pairwise interactions or coupling between gammaGlu-176 and Lys-27 of NmmI and progressively weaker interactions with Arg-33 and Arg-36 in loop II of this three-loop toxin. Thus, loop II of NmmI, and in particular the face of this loop closest to loop III, appears to come into close apposition with Glu-176 of the gamma subunit surface of the binding site interface.