Donor-Dependent and Other Nondefined Factors Have Greater Influence on the Hepatic Phenotype Than the Starting Cell Type in Induced Pluripotent Stem Cell Derived Hepatocyte-Like Cells.

Drug-induced liver injury is the greatest cause of post-marketing drug withdrawal; therefore, substantial resources are directed toward triaging potentially dangerous new compounds at all stages of drug development. One of the major factors preventing effective screening of new compounds is the lack of a predictive in vitro model of hepatotoxicity. Primary human hepatocytes offer a metabolically relevant model for which the molecular initiating events of hepatotoxicity can be examined; however, these cells vary greatly between donors and dedifferentiate rapidly in culture. Induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells (HLCs) offer a reproducible, physiologically relevant and genotypically normal model cell; however, current differentiation protocols produce HLCs with a relatively immature phenotype. During the reprogramming of somatic cells, the epigenome undergoes dramatic changes; however, this "resetting" is a gradual process, resulting in an altered differentiation propensity, skewed toward the lineage of origin, particularly in early passage cultures. We, therefore, performed a comparison of human hepatocyte- and dermal fibroblast-derived iPSCs, assessing the impact of epigenetic memory at all stages of HLC differentiation. These results provide the first isogenic assessment of the starting cell type in human iPSC-derived HLCs. Despite a trend toward improvement in hepatic phenotype in albumin secretion and gene expression, few significant differences in hepatic differentiation capacity were found between hepatocyte and fibroblast-derived iPSCs. We conclude that the donor and inter-clonal differences have a greater influence on the hepatocyte phenotypic maturity than the starting cell type. Therefore, it is not necessary to use human hepatocytes for generating iPSC-derived HLCs. Stem Cells Translational Medicine 2017;6:1321-1331.

Stem cell-derived hepatocytes as a predictive model for drug-induced liver injury: are we there yet?

Amongst the different types of adverse drug reactions, drug-induced liver injury is the most prominent cause of patient morbidity and mortality. However, the current available hepatic model systems developed for evaluating safety have limited utility and relevance as they do not fully recapitulate a fully functional hepatocyte, and do not sufficiently represent the genetic polymorphisms present in the population. The rapidly advancing research in stem cells raises the possibility of using human pluripotent stem cells in bridging this gap. The generation of human induced pluripotent stem cells via reprogramming of mature human somatic cells may also allow for disease modelling in vitro for the purposes of assessing drug safety and toxicology. This would also allow for better understanding of disease processes and thus facilitate in the potential identification of novel therapeutic targets. This review will focus on the current state of effort to derive hepatocytes from human pluripotent stem cells for potential use in hepatotoxicity evaluation and aims to provide an insight as to where the future of the field may lie.

Silicon analogues of the nonpeptidic GnRH antagonist AG-045572: syntheses, crystal structure analyses, and pharmacological characterization.

AG-045572 (CMPD1, 1 a) is a nonpeptidic gonadotropin-releasing hormone (GnRH) antagonist that has been investigated for the treatment of sex hormone-related diseases. In the context of systematic studies on sila-substituted drugs, the silicon analogue disila-AG-045572 (1 b) and its derivative 2 were prepared in multi-step syntheses and characterized by elemental analyses (C, H, N), NMR spectroscopic studies (1H, 13C, 29Si), and single-crystal X-ray diffraction. The pharmacological properties of compounds 1 a, 1 b, and 2 were compared in terms of their in vitro potency at cloned human and rat GnRH receptors. Compounds 1 a and 2 were also examined in regard to their pharmacokinetics and in vivo efficacy in both castrated rat (luteinizing hormone (LH) suppression) and intact rat (testosterone suppression) models. The efficacy and pharmacokinetic profiles of 1 a and its silicon-containing analogue 2 appear similar, indicating that replacement of the 5,6,7,8-tetrahydronaphthalene ring system by the 1,3-disilaindane skeleton led to retention of efficacy. Therefore, the silicon compound 2 represents a novel drug prototype for the design of potent, orally available GnRH antagonists suitable for once-daily dosing.

Neutrophil formylpeptide receptor single nucleotide polymorphism 348T>C in aggressive periodontitis.

BACKGROUND: Neutrophil formylpeptide receptors (FPRs) play an important role in bacterial recognition and chemotaxis. Defective FPR1 expression and impaired polymorphonuclear leukocyte chemotaxis toward bacterial formylpeptides are associated with aggressive periodontitis (AgP). The objective of this study was to determine whether single nucleotide polymorphisms (SNPs) in FPR1 are associated with AgP. METHODS: Genomic DNA was isolated from blood samples obtained from African Americans (30 subjects with AgP and 33 healthy controls) and Turks (75 subjects with AgP and 63 healthy controls). A highly polymorphic fragment of the FPR1 gene was amplified by polymerase chain reaction and sequenced for analysis of SNPs. RESULTS: Five previously identified SNPs were detected in African Americans, whereas six were detected in Turkish subjects. The frequency of synonymous SNP c.348T>C was significantly higher in African Americans with AgP than in controls (P = 0.029). The 348T allele was significantly associated with AgP (P = 0.010). Seven of the subjects with AgP, but none of the controls, were homozygous for 348T. FPR1 haplotypes 348T.568A, 348T.576T, and 348T.568A.576T were significantly increased in African Americans with AgP (P <0.02). The Turkish population did not exhibit significant differences in FPR1 SNP frequencies between subjects with AgP and controls. CONCLUSIONS: FPR1 SNP c.348T>C is associated with AgP in African Americans. Individuals who are homozygous for 348T may have an increased risk for developing this disorder.

Action of (R)-sila-venlafaxine and reboxetine to antagonize cisplatin-induced acute and delayed emesis in the ferret.

The chemotherapeutic drug cisplatin is associated with severe gastrointestinal toxicity that can last for several days. A recent strategy to treat the nausea and emesis includes the combination of a 5-HT3 receptor antagonist, a glucocorticoid, and an NK1 receptor antagonist. The present studies explore the use of the selective noradrenaline reuptake inhibitors, (R)-sila-venlafaxine, (R,R)-reboxetine and (S,S)-reboxetine to prevent cisplatin (5 mg/kg, i.p.)-induced acute (0-24 h) and delayed (24-72 h) emesis in ferrets. The positive control regimen of ondansetron and dexamethasone, both at 1 mg/kg/8 h, reduced acute and delayed emesis by 100 (P<0.001) and 61% (P<0.05). (R)-sila-venlafaxine at 5 and 15 mg/kg/4 h reduced acute emesis by 86 (P<0.01) and 66% (P<0.05), respectively and both enantiomers of reboxetine at 1 mg/kg/12 h also reduced the response by approximately 70-90% (P<0.05). Out of the reuptake inhibitors, only (R)-sila-venlafaxine at 15 mg/kg/4 h was active to reduce delayed emesis (a 57% reduction was observed (P<0.05)); its terminal plasma levels were positively correlated with an inhibition of emesis during the delayed phase (P<0.05). (R)-sila-venlafaxine was also examined against a higher dose of cisplatin 10 mg/kg, i.p. (3 h test) and it dose-dependently antagonized the response (maximum reduction was 94% at 10 mg/kg, p.o.; P<0.01) but it was ineffective against apomorphine (0.125 mg/kg, s.c.) and ipecacuanha (2 mg/kg, p.o.)-induced emesis (P>0.05). In conclusion, the studies provide the first evidence for an anti-emetic potential of noradrenaline reuptake inhibitors to reduce chemotherapy-induced acute and delayed emesis.

Variable responses of formyl peptide receptor haplotypes toward bacterial peptides.

The chemoattractant neutrophil formyl peptide receptor (FPR) binds bacterial and mitochondrial N-formylated peptides, which allows the neutrophils to find the bacterial source and/or site of tissue damage. Certain inflammatory disorders may be due in part to an impaired innate immune system that does not respond to acute bacterial damage in a timely fashion. Because the human FPR is encoded by a large number of different haplotypes arising from ten single-nucleotide polymorphisms, we examined the possibility that some of these haplotypes are functionally distinct. We analyzed the response of three common FPR haplotypes to peptides from Escherichia coli, Mycobacterium avium ssp. paratuberculosis, and human mitochondria. All three haplotypes responded similarly to the E. coli and mitochondrial peptides, whereas one required a higher concentration of the M. avium peptide fMFEDAVAWF for receptor downregulation, receptor signaling, and chemotaxis. This raises the possibility of additional bacterial species differences in functional responses among FPR variants and establishes a precedent with potentially important implications for our innate immune response against bacterial infections. We also investigated whether certain FPR haplotypes are associated with rheumatoid arthritis (RA) by sequencing FPR1 from 148 Caucasian individuals. The results suggested that FPR haplotypes do not significantly contribute toward RA.

Differential activation of polymorphisms of the formyl peptide receptor by formyl peptides.

We have investigated the role of two polymorphic sites (R190W and N192K) on the binding and activation of the formyl peptide receptor (FPR) by viral and formyl peptides. WEDWVGWI, a peptide with antiviral activity derived from the membrane proximal region of feline immunodeficiency virus, binds with high affinity to FPR. The three tryptophans in the peptide are all essential for FPR binding, just as they were essential for antiviral activity [S. Giannecchini, A. Di Fenza, A.M. D'Ursi, D. Matteucci, P. Rovero, M. Bendinelli, Antiviral activity and conformational features of an octapeptide derived from the membrane-proximal ectodomain of the feline immunodeficiency virus transmembrane glycoprotein, J. Virol. 77 (2003) 3724]. Formyl-NleWEDWVGWI behaved as a weak partial agonist with FPR W190/N192 but a stronger partial agonist with FPR R190/K192 and FPR R190/N192. Formyl-NleNleWEDWVGWI behaved as a full agonist toward all three FPRs but exhibited a much higher EC(50) with W190/N192 FPR (300+/-45 nM) than for R190/K192 FPR (40+/-3 nM) or R190/N192 (60+/-8 nM). Formyl-MYKWPWYVWL preferentially activated R190/K192 and R190/N192 FPRs by>5 fold compared to W190/N192 FPR. Formyl-MFEDAVAWF, a peptide derived from a protein in Mycobacterium avium subsp. paratuberculosis and formyl-MFTFEPFPTN, a peptide derived from the N-terminus of chemotaxis inhibitory protein of Staphylococcus aureus with an added N-terminal formyl-methionine exhibited the greatest selectivity for R190/K192 and R190/N192 FPRs with approximately 10 fold lower EC(50)s than that observed with FPR W190/N192. Thus, individuals with the W190 polymorphism may display a reduced ability to detect certain formyl peptides.

Trimethylsilylpyrazoles as novel inhibitors of p38 MAP kinase: a new use of silicon bioisosteres in medicinal chemistry.

The synthesis, physicochemical properties and pharmacological profiles of two novel silicon-containing p38 MAP kinase inhibitors are described.

Peptides derived from HIV-1, HIV-2, Ebola virus, SARS coronavirus and coronavirus 229E exhibit high affinity binding to the formyl peptide receptor.

Peptides derived from the membrane proximal region of fusion proteins of human immunodeficiency viruses 1 and 2, Coronavirus 229 E, severe acute respiratory syndrome coronavirus and Ebola virus were all potent antagonists of the formyl peptide receptor expressed in Chinese hamster ovary cells. Binding of viral peptides was affected by the naturally occurring polymorphisms at residues 190 and 192, which are located at second extracellular loop-transmembrane helix 5 interface. Substitution of R190 with W190 enhanced the affinity for a severe acute respiratory syndrome coronavirus peptide 6 fold but reduced the affinity for N-formyl-Nle-Leu-Phe by 2.5 fold. A 12 mer peptide derived from coronavirus 229E (ETYIKPWWVWL) was the most potent antagonist of the formyl peptide receptor W190 with a K(i) of 230 nM. Fluorescently labeled ETYIKPWWVWL was effectively internalized by all three variants with EC(50) of approximately 25 nM. An HKU-1 coronavirus peptide, MYVKWPWYVWL, was a potent antagonist but N-formyl-MYVKWPWYVWL was a potent agonist. ETYIKPWWVWL did not stimulate GTPgammaS binding but inhibited the stimulation by formyl-NleLeuPhe. It also blocked beta arrestin translocation and receptor downregulation induced by formyl-Nle-Leu-Phe. This indicates that formyl peptide receptor may be important in viral infections and that variations in its sequence among individuals may affect their likelihood of viral and bacterial infections.

(R)-sila-venlafaxine: a selective noradrenaline reuptake inhibitor for the treatment of emesis.

Sila-substitution of drugs (the carbon/silicon switch) is a concept that is being successfully used for the development of new chemical entities. The (R)-sila-analogue of the antidepressant venlafaxine is devoid of the serotonin reuptake inhibition observed with the marketed drug, leading to a selective noradrenaline reuptake inhibitor displaying anti-emetic properties.

Exploitation of silicon medicinal chemistry in drug discovery.

There remains considerable pressure on the pharmaceutical industry to increase productivity and reduce the attrition of drug candidates. Genomics, parallel chemistry and high-throughput biology have not yielded the anticipated benefits, resulting in a renewed focus on validated targets and an aim to generate drugs directed towards such targets, which have a clear advantage. One strategy to identify and develop best-in-class drugs is to apply a high degree of innovation in chemistry and apply this to targets from gene families that have been clinically validated as tractable and drugable. The application of organosilicon medicinal chemistry in the context of privileged structures to aid drug design and development is one such innovative approach that is reviewed in this paper.

Functional epitope on human neutrophil flavocytochrome b558.

mAb NL7 was raised against purified flavocytochrome b(558), important in host defense and inflammation. NL7 recognized the gp91(phox) flavocytochrome b(558) subunit by immunoblot and bound to permeabilized neutrophils and neutrophil membranes. Epitope mapping by phage display analysis indicated that NL7 binds the (498)EKDVITGLK(506) region of gp91(phox). In a cell-free assay, NL7 inhibited in vitro activation of the NADPH oxidase in a concentration-dependent manner, and had marginal effects on the oxidase substrate Michaelis constant (K(m)). mAb NL7 did not inhibit translocation of p47(phox), p67(phox), or Rac to the plasma membrane, and bound its epitope on gp91(phox) independently of cytosolic factor translocation. However, after assembly of the NADPH oxidase complex, mAb NL7 bound the epitope but did not inhibit the generation of superoxide. Three-dimensional modeling of the C-terminal domain of gp91(phox) on a corn nitrate reductase template suggests close proximity of the NL7 epitope to the proposed NADPH binding site, but significant separation from the proposed p47(phox) binding sites. We conclude that the (498)EKDVITGLK(506) segment resides on the cytosolic surface of gp91(phox) and represents a region important for oxidase function, but not substrate or cytosolic component binding.

Chemistry challenges in lead optimization: silicon isosteres in drug discovery.

During the lead optimization phase of drug discovery projects, the factors contributing to subsequent failure might include poor portfolio decision-making and a sub-optimal intellectual property (IP) position. The pharmaceutical industry has an ongoing need for new, safe medicines with a genuine biomedical benefit, a clean IP position and commercial viability. Inherent drug-like properties and chemical tractability are also essential for the smooth development of such agents. The introduction of bioisosteres, to improve the properties of a molecule and obtain new classes of compounds without prior art in the patent literature, is a key strategy used by medicinal chemists during the lead optimization process. Sila-substitution (C/Si exchange) of existing drugs is an approach to search for new drug-like candidates that have beneficial biological properties and a clear IP position. Some of the fundamental differences between carbon and silicon can lead to marked alterations in the physicochemical and biological properties of the silicon-containing analogues and the resulting benefits can be exploited in the drug design process.

Mutations of F110 and C126 of the formyl peptide receptor interfere with G-protein coupling and chemotaxis.

BACKGROUND: Localized aggressive periodontitis (LAgP) is a disease characterized by rapid loss of alveolar bone in teeth of otherwise healthy patients. Neutrophils from LAgP patients have been shown to exhibit diminished chemotaxis and low levels of formyl peptide receptor (FPR) surface expression. A recent study has associated LAgP with 2 polymorphisms in the FPR: 110Phe-->Ser and 126Cys-->Trp. METHODS: We transfected Chinese hamster ovary cells with wtFPR, FPR-110Phe-->Ser, FPR-126Cys-->Trp, or FPR-110Phe-->Ala and determined their surface expression of FPR, their ligand binding affinity, their G-protein coupling, and their chemotaxis toward N-formyl-methionyl-leucyl-phenylalanine (FMLP). RESULTS: FPR-110Phe-->Ser mutants failed to show any significant surface expression or chemotaxis toward FMLP. FPR-126Cys-->Trp mutants exhibited slightly lower than normal binding affinity, markedly lower G-protein coupling response, and markedly lower chemotaxis toward FMLP than that observed with wtFPR. We also analyzed another FPR-Phe110 mutant, FPR-110Phe-->Ala, to ascertain what the effect of mutating this residue might be in a mutant that could be expressed on the cell surface. The FPR-110Phe-->Ala mutant demonstrated markedly lower surface expression, normal ligand binding affinity, markedly lower G-protein coupling, and markedly lower chemotaxis toward FMLP. CONCLUSIONS: Our data substantiate the hypothesis that the chemotactic defects observed in LAgP patients are due at least in part to molecular alterations in the FPR. The FPR-110Phe-->Ser polymorphism appears to be more defective than the FPR-126Cys-->Trp polymorphism, indicating that patients with the former polymorphism might be expected to exhibit a more severe form of aggressive periodontitis.