Duchenne Muscular Dystrophy (DMD) is an X-linked disease characterised by chronic inflammation, progressive muscle damage, and muscle loss. Typically, initial symptoms affect lower limb muscles, including the gastrocnemius (GA), tibialis anterior (TA), and extensor digitorum longus (EDL). During the acute phase of DMD, particularly in boys aged 2–8 years, muscle damage resulting in necrosis (myonecrosis) involves a complex immune-inflammatory response. Prostaglandin D2 (PGD2) is recognised for enhancing pro-inflammatory chemokine and interleukin signalling and recruiting infiltrating immune cells such as pro-inflammatory macrophages, exacerbating myonecrosis. To reduce levels of PGD2, a novel hematopoietic prostaglandin D2 synthase (HPGDS) inhibitor, PK007, was characterised (i) for potency and pharmacokinetic profiles and then tested in the mdx mouse model of DMD during the acute early onset of disease progression. Juvenile mdx and wild type (WT) C57Bl/10Scsn mice were orally treated with PK007 and control vehicle solution for 10 days, from postnatal day 18 to 28. This builds upon a previous study with PK007 with (ii) additional analyses of disease progression assessed for muscle grip strength, metabolic and locomotor activity, myonecrosis in a wide range of muscles (3 from hindlimb, diaphragm, heart, and tongue), macrophage infiltration and pro-inflammatory cytokines (TNF-α, IL-1β and iNOS). PK007 exhibited high potency (17.23 ± 12 nM), a long half-life (3.0 ± 0.3 h), and good oral bioavailability (81

The global burden of neurological disorders is evident, yet there remains limited efficacious therapeutics for their treatment. There is a growing recognition of the role of inflammation in diseases of the central nervous system (CNS); among the numerous inflammatory mediators involved, prostaglandins play a crucial role. Prostaglandins are small lipid mediators derived from arachidonic acid via multi-enzymatic pathways. The actions of prostaglandins are varied, with each prostaglandin having a specific role in maintaining homeostasis. In the CNS, prostaglandins can have neuroprotective or neurotoxic properties depending on their specific G-protein receptor. These G-protein receptors have varying subfamilies, tissue distribution, and signal transduction cascades. Further studies into the impact of prostaglandins in CNS-based diseases may contribute to the clarification of their actions, hopefully leading to the development of efficacious therapeutic strategies. This review focuses on the roles played by prostaglandins in neural degeneration, with a focus on Alzheimer's Disease, Multiple Sclerosis, and Amyotrophic Lateral Sclerosis in both preclinical and clinical settings. We further discuss current prostaglandin-related agonists and antagonists concerning suggestions for their use as future therapeutics.

Abstract Prostaglandin D2 (PGD2) signals via the DP1 and DP2 receptors. In Phase II trials, DP2 antagonism decreased airway inflammation and airway smooth muscle (ASM) area in moderate-to-severe asthma patients. However, in Phase III, DP2 antagonism failed to lower the rate of exacerbations, and DP2 as a target was shelved. Here, using a preclinical model of chronic experimental asthma, we demonstrate that rhinovirus-induced exacerbations increase PGD2 release, mucus production, transforming growth factor (TGF)-β1 and type-2 inflammation. DP2 antagonism or DP1 agonism ablates these phenotypes, increases epithelial EGF expression and decreases ASM area via increased IFN-γ. In contrast, dual DP1-DP2 antagonism or dual corticosteroid/DP2 antagonism, which attenuates endogenous PGD2, prevented ASM resolution. We demonstrate that DP2 antagonism resolves ASM remodelling via PGD2/DP1-mediated upregulation of IFN-γ expression, and that dual DP2 antagonism/corticosteroid therapy, as often occurred in the human trials, impairs the efficacy of DP2 antagonism by suppressing endogenous PGD2 and IFN-γ production.

Duchenne muscular dystrophy (DMD) is characterized by progressive muscle weakness and wasting due to the lack of dystrophin protein. The acute phase of DMD is characterized by muscle necrosis and increased levels of the pro-inflammatory mediator, prostaglandin D2 (PGD2). Inhibiting the production of PGD2 by inhibiting hematopoietic prostaglandin D synthase (HPGDS) may alleviate inflammation and decrease muscle necrosis. We tested our novel HPGDS inhibitor, PK007, in the mdx mouse model of DMD. Our results show that hindlimb grip strength was two-fold greater in the PK007-treated mdx group, compared to untreated mdx mice, and displayed similar muscle strength to strain control mice (C57BL/10ScSn). Histological analyses showed a decreased percentage of regenerating muscle fibers (~20% less) in tibialis anterior (TA) and gastrocnemius muscles and reduced fibrosis in the TA muscle in PK007-treated mice. Lastly, we confirmed that the DMD blood biomarker, muscle creatine kinase activity, was also reduced by ~50% in PK007-treated mdx mice. We conclude that our HPGDS inhibitor, PK007, has effectively reduced muscle inflammation and fibrosis in a DMD mdx mouse model.

Hepcidin peptidomimetics that are orally stable and systemically active will mark a paradigm change in management of blood disorders that exhibit aberrant iron homeostasis (e.g. hereditary hemochromatosis) and in conditions that can be influenced by modulating stressed iron homeostasis (e.g. polycythemia vera). Hepcidin modulates the iron exporter membrane protein ferroportin and is the master regulator of iron homeostasis in the body. Orally bioavailable "Minihepcidins" have been previously shown to be efficacious in lowering serum iron in mice when dosed peroral (PO) (Preza GC et. al., Journal of Clinical Investigation 2011). Here we describe hepcidin mimetic peptides that are metabolically stable in the gastrointestinal tract, systemically absorbed when delivered orally, and pharmacodynamically active in reducing serum iron parameters in pre-clinical models. Further, we also demonstrate improvement in disease parameters in a mouse model for hereditary hemochromatosis. The oral peptides, PN20076 and PN20089, have EC50 of 16.5 nM and 1.39 nM respectively in cell based ferroportin internalization assay (Table 1). In comparison EC50 was 67.8 nM for Hepcidin and 6.12 nM for PTG-300. (PTG-300 is an injectable hepcidin mimetic currently in Phase 2 clinical studies for polycythemia vera and hereditary hemochromatosis.) Oral stability of the peptides was evaluated in a panel of assays, including in vitro matrices simulating the gastric and intestinal conditions, and ex vivo matrices of serum/plasma from different species. Table 1 shows data for peptides PN20018, PN20076 and PN20089. PN20076 demonstrated extended stability in gastric and intestinal conditions, and degradation half-life of >24 hr in mouse plasma and 14.8 hr in rat serum. Based on their stability and potency data from the above battery of screening assays, the peptides were selected for in vivo evaluation in healthy mice to characterize their pharmacodynamic (PD) and pharmacokinetic (PK) properties. PN20076 and PN20089 showed equivalent PD response of reduction in serum iron concentration in wild type mice. After two successive PO doses of PN20076 or PN20089 approximately 24 hr apart, serum iron concentration was reduced from ~30 µM to ~10 µM (group averages), i.e. ~66% reduction, at 4.5 hr post-second dose for both peptides (Fig. 1). At 4.5 hr post-dose, the serum concentration of PN20076 was ~262 nM. PN20076 was further evaluated for its effect in lowering iron overload in a mouse model for hemochromatosis (HFE2-/- with homozygous deletion of hemojuvelin, a positive regulator of hepcidin expression). This mouse model is marked by hyper-absorption of dietary iron, higher transferrin saturation and deposition of excessive iron in liver, all manifestations of aberrant iron homeostasis caused by the genetic disruptions of the hepcidin-iron pathway. Liver iron accumulation was significantly prevented in groups treated with PN20076 once daily (QD) by PO administration for over two weeks, as compared to vehicle treated controls (Fig. 2). The reduction in non-heme iron concentration in liver homogenates (measured using a colorimetric iron assay) was statistically significant in the female group treated with PN20076. We have described orally stable and systemically active hepcidin mimetic peptides and demonstrated oral activity in preventing liver iron overload in hemochromatosis mice. The effective reduction of iron absorption from the diet and the steady state lowering of transferrin-saturation can potentially prevent tissue iron toxicity in hereditary hemochromatosis. Similarly, the sustained reduction of systemic iron levels with an oral hepcidin mimetic to control stressed iron homeostasis should reduce excessive erythrocytosis, a hallmark of polycythemia vera and other congenital and acquired erythropoietic disorders. Disclosures Bourne: Protagonist Therapeutics: Current Employment, Other: shareholder. Zhang:Protagonist Therapeutics: Current Employment, Other: shareholder. Frederick:Protagonist Therapeutics: Current Employment, Other: shareholder. Tran:Protagonist Therapeutics: Current Employment, Other: shareholder. Vengalam:Protagonist Therapeutics: Current Employment, Current equity holder in private company. McMahon:Protagonist Therapeutics: Current Employment, Other: shareholder. Huie:Protagonist Therapeutics: Current Employment, Other: shareholder. Ledet:Protagonist Therapeutics: Current Employment, Other: shareholder. Zhao:Protagonist Therapeutics: Current Employment, Other: shareholder. Tovera:Protagonist Therapeutics: Current Employment, Current equity holder in private company. Lee:Protagonist Therapeutics: Current Employment, Current equity holder in private company. Yang:Protagonist Therapeutics: Current Employment, Other: shareholder. Dion:Protagonist Therapeutics: Current Employment, Current equity holder in private company. Yuan:Protagonist Therapeutics: Current Employment, Other: shareholder. Zemede:Protagonist Therapeutics: Current Employment, Current equity holder in private company. Nguyen:Protagonist Therapeutics: Current Employment, Current equity holder in private company. Masjedizadeh:Protagonist Therapeutics: Current Employment, Current equity holder in private company. Cheng:Protagonist Therapeutics: Current Employment, Current equity holder in private company. Mattheakis:Protagonist Therapeutics: Current Employment, Current equity holder in private company. Liu:Protagonist Therapeutics: Current Employment, Current equity holder in private company. Smythe:Protagonist Therapeutics: Current Employment, Other: shareholder.

Prostaglandin D2 (PGD2) signals through PGD2 receptor 2 (DP2, also known as CRTH2) on type 2 effector cells to promote asthma pathogenesis; however, little is known about its role during respiratory syncytial virus (RSV) bronchiolitis, a major risk factor for asthma development. We show that RSV infection up-regulated hematopoietic prostaglandin D synthase expression and increased PGD2 release by cultured human primary airway epithelial cells (AECs). Moreover, PGD2 production was elevated in nasopharyngeal samples from young infants hospitalized with RSV bronchiolitis compared to healthy controls. In a neonatal mouse model of severe viral bronchiolitis, DP2 antagonism decreased viral load, immunopathology, and morbidity and ablated the predisposition for subsequent asthma onset in later life. This protective response was abolished upon dual DP1/DP2 antagonism and replicated with a specific DP1 agonist. Rather than mediating an effect via type 2 inflammation, the beneficial effects of DP2 blockade or DP1 agonism were associated with increased interferon-λ (IFN-λ) [interleukin-28A/B (IL-28A/B)] expression and were lost upon IL-28A neutralization. In RSV-infected AEC cultures, DP1 activation up-regulated IFN-λ production, which, in turn, increased IFN-stimulated gene expression, accelerating viral clearance. Our findings suggest that DP2 antagonists or DP1 agonists may be useful antivirals for the treatment of viral bronchiolitis and possibly as primary preventatives for asthma.

Background Disulfide-rich peptides (DRPs) are found throughout nature. They are suitable scaffolds for drug development due to their small cores, whose disulfide bonds impart extraordinary chemical and biological stability. A challenge in developing a DRP therapeutic is to engineer binding to a specific target. This challenge can be overcome by (i) sampling the large sequence space of a given scaffold through a phage display library and by (ii) panning multiple libraries encoding structurally distinct scaffolds. Here, we implement a protocol for defining these diverse scaffolds, based on clustering structurally defined DRPs according to their conformational similarity. Results We developed and applied a hierarchical clustering protocol based on DRP structural similarity, followed by two post-processing steps, to classify 806 unique DRP structures into 81 clusters. The 20 most populated clusters comprised 85% of all DRPs. Representative scaffolds were selected from each of these clusters; the representatives were structurally distinct from one another, but similar to other DRPs in their respective clusters. To demonstrate the utility of the clusters, phage libraries were constructed for three of the representative scaffolds and panned against interleukin-23. One library produced a peptide that bound to this target with an IC 50 of 3.3 μM. Conclusions Most DRP clusters contained members that were diverse in sequence, host organism, and interacting proteins, indicating that cluster members were functionally diverse despite having similar structure. Only 20 peptide scaffolds accounted for most of the natural DRP structural diversity, providing suitable starting points for seeding phage display experiments. Through selection of the scaffold surface to vary in phage display, libraries can be designed that present sequence diversity in architecturally distinct, biologically relevant combinations of secondary structures. We supported this hypothesis with a proof-of-concept experiment in which three phage libraries were constructed and panned against the IL-23 target, resulting in a single-digit μM hit and suggesting that a collection of libraries based on the full set of 20 scaffolds increases the potential to identify efficiently peptide binders to a protein target in a drug discovery program.

Interleukin-6 (IL-6) is an important member of the cytokine superfamily, exerting pleiotropic actions on many physiological processes. Over-production of IL-6 is a hallmark of immune-mediated inflammatory diseases such as Castleman's Disease (CD) and rheumatoid arthritis (RA). Antagonism of the interleukin IL-6/IL-6 receptor (IL-6R)/gp130 signaling complex continues to show promise as a therapeutic target. Monoclonal antibodies (mAbs) directed against components of this complex have been approved as therapeutics for both CD and RA. To potentially provide an additional modality to antagonize IL-6 induced pathophysiology, a peptide-based antagonist approach was undertaken. Using a combination of molecular design, phage-display, and medicinal chemistry, disulfide-rich peptides (DRPs) directed against IL-6 were developed with low nanomolar potency in inhibiting IL-6-induced pSTAT3 in U937 monocytic cells. Targeted PEGylation of IL-6 binding peptides resulted in molecules that retained their potency against IL-6 and had a prolongation of their pharmacokinetic (PK) profiles in rodents and monkeys. One such peptide, PN-2921, contained a 40 kDa polyethylene glycol (PEG) moiety and inhibited IL-6-induced pSTAT3 in U937 cells with sub-nM potency and possessed 23, 36, and 59 h PK half-life values in mice, rats, and cynomolgus monkeys, respectively. Parenteral administration of PN-2921 to mice and cynomolgus monkeys potently inhibited IL-6-induced biomarker responses, with significant reductions in the acute inflammatory phase proteins, serum amyloid A (SAA) and C-reactive protein (CRP). This potent, PEGylated IL-6 binding peptide offers a new approach to antagonize IL-6-induced signaling and associated pathophysiology.

Mammalian cytosolic glutathione transferases (GSTs) can be grouped into seven classes. Of these, the sigma class is also widely distributed in nature, with isoforms found in both vertebrates and invertebrates. It contains examples of proteins that have evolved specialized functions, such as the cephalopod lens S-crystallins, the mammalian hematopoietic prostaglandin D2 synthase, and the helminth 28-kDa antigen. In mammals, the sigma-class GST has both anti- and proinflammatory functions, depending on the type of immune response, and an immunomodulatory function is also associated with the enzyme from helminth parasites. In the fly, it is associated with a specific detoxication activity toward lipid oxidation products. Mice genetically depleted of the sigma-class GST, or transgenically overexpressing it, have provided insight into the physiological roles of the GST. Inhibitors of the mammalian enzyme developed by structure-based methods are effective in controlling allergic response. This review covers the structure, function, and pharmacology of vertebrate and invertebrate GSTs.

A new population‐based incremental learning algorithm for conformational searching of molecules is presented. This algorithm is particularly effective at determining, by relatively small number of energy minimizations, global energy minima of large flexible molecules. The algorithm is also able to find a large set of low energy conformations of more rigid small molecules. The performance of the algorithm is relation to other algorithm is examined via the test molecules: C18H38, C39H80, cycloheptadecane and a set of five drug‐like molecules. © 2011 Wiley Periodicals, Inc. J Comput Chem, 2011

The hematopoietic prostaglandin D(2) synthase has a proinflammatory effect in a range of diseases, including allergic asthma, where its product prostaglandin D(2) (PGD(2)) has a role in regulating many of the hallmark disease characteristics. Here we describe the development and characterization of a novel series of hematopoietic prostaglandin D(2) synthase inhibitors with potency similar to that of known inhibitors. Compounds N-benzhydryl-5-(3-hydroxyphenyl)thiophene-2-carboxamide (compound 8) and N-(1-amino-1-oxo-3-phenylpropan-2-yl)-6-(thiophen-2-yl)nicotinamide (compound 34) demonstrated low micromolar potency in the inhibition of the purified enzyme, while only 34 reduced Toll-like receptor (TLR) inducible PGD(2) production in both mouse primary bone marrow-derived macrophages and the human megakaryocytic cell line MEG-01S. Importantly, 34 demonstrated a greater selectivity for inhibition of PGD(2) synthesis versus other eicosanoids that lie downstream of PGH(2) (PGE(2) and markers of prostacyclin (6-keto PGF(1alpha)) and thromboxane (TXB(2))) when compared to the known inhibitors HQL-79 (compound 1) and 2-phenyl-5-(1H-pyrazol-3-yl)thiazole (compound 2). Compound 34 therefore represents a selective hematopoietic prostaglandin D(2) synthase inhibitor.

Medicinal chemists synthesize arrays of molecules by attaching functional groups to scaffolds. There is evidence suggesting that some scaffolds yield biologically active molecules more than others, these are termed privileged substructures. One role of the scaffold is to present its side-chains for molecular recognition, and biologically relevant scaffolds may present side-chains in biologically relevant geometries or shapes. Since drug discovery is primarily focused on the discovery of compounds that bind to proteinaceous targets, we have been deciphering the scaffold shapes that are used for binding proteins as they reflect biologically relevant shapes. To decipher the scaffold architecture that is important for binding protein surfaces, we have analyzed the scaffold architecture of protein loops, which are defined in this context as continuous four residue segments of a protein chain that are not part of an α-helix or β-strand secondary structure. Loops are an important molecular recognition motif of proteins. We have found that 39 clusters reflect the scaffold architecture of 89% of the 23,331 loops in the dataset, with average intra-cluster and inter-cluster RMSD of 0.47 and 1.91, respectively. These protein loop scaffolds all have distinct shapes. We have used these 39 clusters that reflect the scaffold architecture of protein loops as biological descriptors. This involved generation of a small dataset of scaffold-based peptidomimetics. We found that peptidomimetic scaffolds with reported biological activities matched loop scaffold geometries and those peptidomimetic scaffolds with no reported biologically activities did not. This preliminary evidence suggests that organic scaffolds with tight matches to the preferred loop scaffolds of proteins, implies the likelihood of the scaffold to be biologically relevant.

Prostaglandin D(2) synthesised by the hematopoietic prostaglandin D(2) synthase has a pro-inflammatory effect in allergic asthma, regulating many hallmark characteristics of the disease. Here we describe identification of hematopoietic prostaglandin D(2) synthase inhibitors including cibacron blue, bromosulfophthalein and ethacrynic acid. Expansion around the drug-like ethacrynic acid identified a novel inhibitor, nocodazole, and a fragment representing its aromatic core. Nocodazole binding was further characterised by docking calculations in combination with conformational strain analysis. The benzyl thiophene core was predicted to be buried in the active site, binding in the putative prostaglandin binding site, and a likely hydrogen bond donor site identified. X-ray crystallographic studies supported the predicted binding mode.