Design and synthesis of biologically active peptides: a 'tail' of amino acids can modulate activity of synthetic cyclic peptides

In earlier work, we synthesized a cyclic 9-amino acid peptide (AFPep, cyclo[EKTOVNOGN]) and showed it to be useful for prevention and therapy of breast cancer. In an effort to explore the structure–function relationships of AFPep, we have designed analogs that bear a short ‘tail’ (one or two amino acids) attached to the cyclic peptide distal to its pharmacophore. Analogs that bore a tail of either one or two amino acids, either of which had a hydrophilic moiety in the side chain (e.g., cyclo[EKTOVNOGN]FS) exhibited greatly diminished biological activity (inhibition of estrogen-stimulated uterine growth) relative to AFPep. Analogs that bore a tail of either one or two amino acids which had hydrophobic (aliphatic or aromatic) side chains (e.g., cyclo[EKTOVNOGN]FI) retained (or had enhanced) growth inhibition activity. Combining in the same biological assay a hydrophilic-tailed analog with either AFPep or a hydrophobic-tailed analog resulted in decreased activity relative to that for AFPep or for the hydrophobic-tailed analog alone, suggesting that hydrophilic-tailed analogs are binding to a biologically active receptor. An analog with a disrupted pharmacophore (cyclo[EKTOVGOGN]) exhibited little or no growth inhibition activity. An analog with a hydrophilic tail and a disrupted pharmacophore (cyclo[EKTOVGOGN]FS) exhibited no growth inhibition activity of its own and did not affect the activity of a hydrophobic-tailed analog, but enhanced the growth inhibition activity of AFPep. These results are discussed in the context of a two-receptor model for binding of AFPep and ring-and-tail analogs. We suggest that tails on cyclic peptides may comprise a useful method to enhance diversity of peptide design and specificity of ligand–receptor interactions.     

Small molecule antagonists of CCR8 inhibit eosinophil and T cell migration

In this study, we demonstrate that in addition to T lymphocytes, human naïve eosinophils and the differentiated eosinophil-like cell line, AML14.3D10 express CCR8 and respond to CCL1 through CCR8 engagement. The responsiveness of cells was dependent on maturation stage, since CCL1 induced pronounced chemotaxis only in differentiated CCR8 positive AML14.3D10 cells. Despite the low CCR8 surface expression, human naïve eosinophils respond with a chemotaxis to high concentration CCL1. We further describe that Th2 clones in a maturation dependent fashion produce autocrine CCL1, which renders them unresponsive to further stimulation. An innovative method to enrich primary CCR8 reactive T cells was developed which demonstrates that primary peripheral CCR8 expressing T cells respond significantly to CCL1.We have developed novel small molecule CCR8 antagonists that are effective in inhibiting calcium mobilization and chemotaxis in differentiated AML cells as well as in human primary CCR8 positive T cells. Importantly, we demonstrate that the compounds can be divided into two subgroups: (i) compounds that are functional agonists for calcium mobilization and chemotaxis (ii) compounds that are pure antagonists. We demonstrate that agonism of these compounds does not correlate with their antagonistic potency. Taken together, we have identified a novel set of CCR8 compounds with antagonistic properties that inhibit CCL1 driven chemotaxis in both CCR8 expressing eosinophils as well as primary human T cells.     

Characterization of sparstolonin B, a Chinese herb-derived compound, as a selective Toll-like receptor antagonist with potent anti-inflammatory properties

Blockade of excessive Toll-like receptor (TLR) signaling is a therapeutic approach being actively pursued for many inflammatory diseases. Here we report a Chinese herb-derived compound, sparstolonin B (SsnB), which selectively blocks TLR2- and TLR4-mediated inflammatory signaling. SsnB was isolated from a Chinese herb, Spaganium stoloniferum; its structure was determined by NMR spectroscopy and x-ray crystallography. SsnB effectively inhibited inflammatory cytokine expression in mouse macrophages induced by lipopolysaccharide (LPS, a TLR4 ligand), Pam3CSK4 (a TLR1/TLR2 ligand), and Fsl-1 (a TLR2/TLR6 ligand) but not that by poly(I:C) (a TLR3 ligand) or ODN1668 (a TLR9 ligand). It suppressed LPS-induced cytokine secretion from macrophages and diminished phosphorylation of Erk1/2, p38a, IκBα, and JNK in these cells. In THP-1 cells expressing a chimeric receptor CD4-TLR4, which triggers constitutive NF-κB activation, SsnB effectively blunted the NF-κB activity. Co-immunoprecipitation showed that SsnB reduced the association of MyD88 with TLR4 and TLR2, but not that with TLR9, in HEK293T cells and THP-1 cells overexpressing MyD88 and TLRs. Furthermore, administration of SsnB suppressed splenocyte inflammatory cytokine expression in mice challenged with LPS. These results demonstrate that SsnB acts as a selective TLR2 and TLR4 antagonist by blocking the early intracellular events in the TLR2 and TLR4 signaling. Thus, SssB may serve as a promising lead for the development of selective TLR antagonistic agents for inflammatory diseases.     

Identifying structural features on 1,1-diphenyl-hexahydro-oxazolo[3,4-a]pyrazin-3-ones critical for Neuropeptide S antagonist activity

A series of 7-substituted 1,1-diphenyl-hexahydro-oxazolo[3,4-a]pyrazin-3-ones were synthesized and tested for Neuropeptide S (NPS) antagonist activity. A concise synthetic route was developed, which features a DMAP catalyzed carbamate formation. 4-Fluorobenzyl urea (1c) and benzyl urea (1d) were identified as the most potent antagonists among the compounds examined. Structure-activity relationships (SARs) demonstrate that a 7-position urea functionality is required for potent antagonist activity and alkylation of the urea nitrogen (1e) or replacement with carbon or oxygen (5a) results in reduced potency. In addition, compounds with alpha-methyl substitution (1b) or elongated alkyl chains (1h and 1j) had reduced potency, indicating a limited tolerance for 7-position substituents.     

Construction and characterization of the HMGB1 mutant as a competitive antagonist to HMGB1 induced cytokines release

Recent studies indicate that the High Mobility Group Box-1 protein (HMGB1) acts as a potent proinflammatory cytokine that contributes to the pathogenesis of diverse inflammatory and infectious disorders. The proinflammatory cytokine activity of HMGB1 has become a therapeutic target. In this study, we cloned the cDNA encoding human HMGB1 and constructed HMGB1 mutants using a one-step opposite direction PCR. The binding of the HMGB1 mutants to THP-1 cell and the cytokine activities of these HMGB1 mutants were observed. Results showed that the HMGB1 Mut (102-105), one of the HMGB1 mutants, in which amino acids 102-105 (FFLF) were replaced with two Glys, significantly decreased the full-length HMGB1 protein induced TNF-α release in human monocyte cultures. The results indicate that we have developed a novel recombinant HMGB1 mutant that competitively antagonizes the proinflammatory activity of HMGB1. This may be of significant importance in providing a new anti-inflammatory strategy for the treatment of severe sepsis and other inflammatory disorders.     

Inhibition of neuropeptide FF (NPFF)-induced hypothermia and anti-morphine analgesia by RF9, a new selective NPFF receptors antagonist

Neuropeptide FF (NPFF) belongs to a neuropeptide family including two precursors (pro-NPFF_A and pro-NPFF_B) and two receptors (NPFF₁ and NPFF₂). Very recently, the novel compound RF9 was reported as the truly selective antagonist on NPFF receptors. The present study examined the effects of RF9 on the hypothermia and anti-morphine action induced by NPFF in mice. (1) RF9 injected into the third ventricle was devoid of any residual agonist activity, but it completely antagonized the hypothermic effects of NPFF (30 or 45 nmol) after cerebral administration in mice; (2) RF9 did not alter the tail-flick latency and morphine analgesia in nociceptive test, however, co-administration of RF9 prevented the anti-morphine action of intracerebroventricularly applied NPFF (10 nmol, i.c.v.) in the mouse tail-flick assay. Collectively, our data indicate that RF9, behaving as a truly pure NPFF receptors antagonist, prevents NPFF-induced drops of the body temperature and morphine analgesia in mice. In addition, it further confirms that the hypothermia and anti-morphine action of NPFF are mediated directly by NPFF receptors.     

Pantolactams as androgen receptor antagonists for the topical suppression of sebum production

A series of pantolactam based compounds were identified as potent antagonists for the androgen receptor (AR). Those that possessed properties suitable for topical delivery were evaluated in the validated Hamster Ear Model. Several compounds were found to be efficacious in reducing wax esters, a major component of sebum, initiating further preclinical work on these compounds.     

Towards rational design of cannabinoid receptor 1 (CB1) antagonists for peripheral selectivity

CB1 receptor antagonists that are peripherally restricted were targeted. Compounds with permanent charge as well as compounds that have increased polar surface area were made and tested against CB1 for binding and activity. Sulfonamide and sulfamide with high polar surface area and good activity at CB1 were rationally designed and pharmacologically tested. Further optimization of these compounds and testing could lead to the development of a new class of therapeutics to treat disorders where the CB1 receptor system has been implicated.     

Discovery of novel pyridyl carboxamides as potent CCR5 antagonists and optimization of their pharmacokinetic profile in rats

A novel series of pyridyl carboxamide-based CCR5 inhibitors was designed, synthesized, and demonstrated to be highly potent against HIV-1 infection in both HOS and PBL assays. Attempts to evaluate this series of compounds in a rat PK model revealed its instability in rat plasma. A hypothesis for this liability was proposed, and strategies to overcome this issue were pursued, leading to discovery of highly potent 40 and 41, which featured dramatically improved rat PK profiles.     

1-(Arylsulfonyl)-2,3-dihydro-1H-quinolin-4-one derivatives as 5-HT(6) serotonin receptor ligands

Piperazinyl derivatives of 1-(arylsulfonyl)-2,3-dihydro-1H-quinolin-4-ones have been identified with high binding affinities for 5-HT₆ receptor. In particular, 2-methyl-5-(N-methyl-piperazin-1-yl)-1-(naphthalene-2-sulfonyl)-2,3-dihydro-1H-quinolin-4-one (8g) exhibits high binding affinity toward 5-HT₆ (IC₅₀ = 8 nM) receptor with good selectivity over other serotonin and dopamine receptors.