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With over 200 million cases and close to half a million deaths each year, malaria is a threat to global health, particularly in developing countries.

Treatment of tuberculosis (TB) currently takes at least 6 months. Latent Mycobacterium tuberculosis (Mtb) is phenotypically tolerant to most anti-TB drugs. A key hypothesis is that drugs that kill nonreplicating (NR) Mtb may shorten treatment when used in combination with conventional drugs. The Mtb proteasome (Mtb20S) could be such a target because its pharmacological inhibition kills NR Mtb and its genetic deletion renders Mtb unable to persist in mice. Here, we report a series of macrocyclic peptides that potently and selectively target the Mtb20S over human proteasomes, including macrocycle 6. The cocrystal structure of macrocycle 6 with Mtb20S revealed structural bases for the species selectivity. Inhibition of 20S within Mtb by 6 dose dependently led to the accumulation of Pup-tagged GFP that is degradable but resistant to depupylation and death of nonreplicating Mtb under nitrosative stress. These results suggest that compounds of this class have the potential to develop as anti-TB therapeutics.

Proteasomes of pathogenic microbes have become attractive targets for anti-infectives. Coevolving with its human host, Mycobacterium tuberculosis (Mtb) has developed mechanisms to resist host-impos...

[Image: see text] Aberrant gene-silencing through dysregulation of polycomb protein activity has emerged as an important oncogenic mechanism in cancer, implicating polycomb proteins as important therapeutic targets. Recently, an inhibitor targeting EZH2, the methyltransferase component of PRC2, received U.S. Food and Drug Administration approval following promising clinical responses in cancer patients. However, the current array of EZH2 inhibitors have poor brain penetrance, limiting their use in patients with central nervous system malignancies, a number of which have been shown to be sensitive to EZH2 inhibition. To address this need, we have identified a chemical strategy, based on computational modeling of pyridone-containing EZH2 inhibitor scaffolds, to minimize P-glycoprotein activity, and here we report the first brain-penetrant EZH2 inhibitor, TDI-6118 (compound 5). Additionally, in the course of our attempts to optimize this compound, we discovered TDI-11904 (compound 21), a novel, highly potent, and peripherally active EZH2 inhibitor based on a 7 member ring structure.

Aberrant gene-silencing through dysregulation of polycomb protein activity has emerged as an important oncogenic mechanism in cancer, implicating polycomb proteins as important therapeutic targets. Recently, an inhibitor targeting EZH2, the methyltransferase component of PRC2, received FDA approval following promising clinical responses in cancer patients. However, the current array of EZH2 inhibitors have poor brain-penetrance limiting their use in patients with CNS malignancies, a number of which have been shown to be sensitive to EZH2 inhibition. To address this need, we have identified a chemical strategy, based on computational modeling of pyridone-containing EZH2 inhibitor scaffolds, to minimize P-glycoprotein activity and here we report the first brain-penetrant EZH2 inhibitor, TDI-6118 (compound 5). Additionally, in the course of our attempts to optimize this compound we discovered TDI-11904 (compound 21); a novel, highly-potent, and peripherally active EZH2 inhibitor based on a 7 member ring structure.

Treatment of tuberculosis (TB) currently takes at least 6 months. Latent Mycobacterium tuberculosis (Mtb) are phenotypically tolerant to most anti-TB drugs. A key hypothesis is that drugs that kill non-replicating (NR) Mtb may shorten treatment when used in combination with conventional drugs. The Mtb proteasome (Mtb20S) could be such a target, because its pharmacological inhibition kills NR Mtb and its genetic deletion renders Mtb unable to persist in mice. Here we report a series of macrocyclic peptides that potently and selectively target the Mtb20S over human proteasomes, including macrocycle 6. The co-crystal structure of macrocycle 6 with Mtb20S revealed structural bases for the species selectivity. Inhibition of 20S within Mtb by 6 dose-dependently led to accumulation of Pup-tagged GFP that is degradable but resistant to depupylation., and death of non-replicating Mtb under nitrosative stress. These results suggest that compounds of this class have the potential to develop as anti-TB therapeutics.

[Image: see text] Tuberculosis remains a leading cause of death from a single bacterial infection worldwide. Efforts to develop new treatment options call for expansion into an unexplored target space to expand the drug pipeline and bypass resistance to current antibiotics. Lipoamide dehydrogenase is a metabolic and antioxidant enzyme critical for mycobacterial growth and survival in mice. Sulfonamide analogs were previously identified as potent and selective inhibitors of mycobacterial lipoamide dehydrogenase in vitro but lacked activity against whole mycobacteria. Here we present the development of analogs with improved permeability, potency, and selectivity, which inhibit the growth of Mycobacterium tuberculosis in axenic culture on carbohydrates and within mouse primary macrophages. They increase intrabacterial pyruvate levels, supporting their on-target activity within mycobacteria. Distinct modalities of binding between the mycobacterial and human enzymes contribute to improved potency and hence selectivity through induced-fit tight binding interactions within the mycobacterial but not human enzyme, as indicated by kinetic analysis and crystallography.

Plasmodium falciparum proteasome (Pf20S) inhibitors are active against Plasmodium at multiple stages - erythrocytic stages, gametocyte stages, liver stages and gamete activation, indicating that selective Pf20S inhibitors possess the potential to be therapeutic, prophylactic and transmission-blocking antimalarials. Starting from a reported compound, we developed a noncovalent, macrocyclic peptide inhibitor of the malarial proteasome with high species selectivity and improved pharmacokinetic properties. The compound demonstrates specific, time-dependent inhibition of the b5 subunit of the Plasmodium falciparum proteasome, kills artemisinin-sensitive and artemisinin-resistant P. falciparum isolates in vitro and reduces parasitemia in humanized, P. falciparum -infected mice.

CCR6 has been implicated in both autoimmune diseases and non-autoimmune diseases. Thus, inhibition of CCR6-dependent cell migration is an attractive strategy for their treatment. An orally available small molecule inhibitor of CCR6 could therefore be a useful biological probe for the pathophysiological studies. Initial SAR study of a hit compound provided potent N-benzenesulfonylpiperidine derivatives that suppressed CCL20-induced Gi signals. By subsequent scaffold morphing of the central ring and further optimization, we identified a novel series of 1,4-trans-1-benzenesulfonyl-4-aminocyclohexanes as potent and selective CCR6 inhibitors with good pharmacokinetic properties. Our compounds showed good correlation between Gi signal inhibitory activity and cell migration inhibitory activity in human CCR6-transfected CHO cells. In addition, representative compound 35 potently inhibited CCR6-dependent cell migration and the increase in ERK phosphorylation in human primary cells. Therefore, the compound could be used effectively as a biological probe against human CCR6.

G-protein-coupled receptor 52 (GPR52) is classified as an orphan Gs-coupled G-protein-coupled receptor. GPR52 cancels dopamine D2 receptor signaling and activates dopamine D1/N-methyl-d-aspartate receptors via intracellular cAMP accumulation. Therefore, GPR52 agonists are expected to alleviate symptoms of psychotic disorders. A novel series of 1-(benzothiophen-7-yl)-1H-pyrazole as GPR52 agonists was designed and synthesized based on compound 1b. Compound 1b has been reported by our group as the first orally active GPR52 agonist, but high lipophilicity and poor aqueous solubility still remained as issues for candidate selection. To resolve these issues, replacement of the benzene ring at the 7-positon of compound 1b with heterocylic rings, such as pyrazole and pyridine, was greatly expected to reduce lipophilicity to levels for which calculated logD values were lower than that of compound 1b. While evaluating the pyrazole derivatives, introduction of a methyl substituent at the 3-position of the pyrazole ring led to increased GPR52 agonistic activity. Moreover, additional methyl substituent at the 5-position of the pyrazole and further introduction of hydroxy group to lower logD led to significant improvement of solubility while maintaining the activity. As a result, we identified 3-methyl-5-hydroxymethyl-1H-pyrazole derivative 17 (GPR52 EC50 = 21 nM, Emax = 103%, logD = 2.21, Solubility at pH 6.8 = 21 μg/mL) with potent GPR52 agonistic activity and good solubility compared to compound 1b. Furthermore, this compound 17 dose-dependently suppressed methamphetamine-induced hyperlocomotion in mice.

Accumulating evidence suggests that fibrinogen, a key protein in the coagulation cascade, plays an important role in circulatory dysfunction in Alzheimer’s disease (AD). Previous work has shown that the interaction between fibrinogen and β-amyloid (Aβ), a hallmark pathological protein in AD, induces plasmin-resistant abnormal blood clots, delays fibrinolysis, increases inflammation, and aggravates cognitive function in mouse models of AD. Since Aβ oligomers have a much stronger affinity for fibrinogen than Aβ monomers, we tested whether amyloid aggregation inhibitors could block the Aβ-fibrinogen interaction and found that some Aβ aggregation inhibitors showed moderate inhibitory efficacy against this interaction. We then modified a hit compound so that it not only showed a strong inhibitory efficacy towards the Aβ-fibrinogen interaction but also retained its potency towards the Aβ42 aggregation inhibition process. Furthermore, our best hit compound, TDI-2760, modulated Aβ42-induced contact system activation, a pathological condition observed in some AD patients, in addition to inhibiting the Aβ-fibrinogen interaction and Aβ aggregation. Thus, TDI-2760 has the potential to lessen vascular abnormalities as well as Aβ aggregation-driven pathology in AD.

The aim of this study was to investigate peripheral and central roles of corticotropin-releasing factor (CRF) in endocrinological and behavioral changes. Plasma adrenocorticotropin (ACTH) concentration was measured as an activity of hypothalamic-pituitary-adrenal (HPA) axis. As behavioral changes, locomotion and anxiety behavior were measured after CRF challenge intravenously (i.v.) for the peripheral administration or intracerebroventricularly (i.c.v.) for the central administration. Plasma ACTH concentration was significantly increased by both administration routes of CRF; however, hyperlocomotion and anxiety behavior were induced only by the i.c.v. administration. In the drug discovery of CRF1 receptor antagonists, we identified two types of compounds, Compound A and Compound B, which antagonized peripheral CRF-induced HPA axis activation to the same extent, but showed different effects on the central CRF signal. These had similar in vitro CRF1 receptor binding affinities (15 and 10nM) and functional activities in reporter gene assay (15 and 9.5nM). In the ex vivo binding assays using tissues of the pituitary, oral treatment with Compound A and Compound B at 10mg/kg inhibited [125I]-CRF binding, whereas in the assay using tissues of the frontal cortex, treatment of Compound A but not Compound B inhibited [125I]-CRF binding, indicating that only Compound A inhibited central [125I]-CRF binding. In the peripheral CRF challenge, increase in plasma ACTH concentration was significantly suppressed by both Compound A and Compound B. In contrast, Compound A inhibited the increase in locomotion induced by the central CRF challenge while Compound B did not. Compound A also reduced central CRF challenge-induced anxiety behavior and c-fos immunoreactivity in the cortex and the hypothalamic paraventricular nucleus. These results indicate that the central CRF signal, rather than the peripheral CRF signal would be related to anxiety and other behavioral changes, and CRF1 receptor antagonism in the central nervous system may be critical for identifying drug candidates for anxiety and mood disorders.

Background The major symptoms of irritable bowel syndrome (IBS) are changes in bowel habits and abdominal pain. Psychological stress is the major pathophysiological components of IBS. Corticotropin-releasing factor (CRF) is a well-known integrator in response to psychological stress. In this study, a novel CRF1 receptor antagonist T-3047928 was evaluated in stress-induced IBS models of rats to explore its potency for IBS. Methods Plasma adrenocorticotropic hormone (ACTH) levels after intravenous oCRH challenge were measured as a pharmacodynamic marker. Efficacies of oral T-3047928 were compared with oral alosetron, a 5-HT3 antagonist, on conditioning fear stress (CFS)-induced defecation, restraint stress (RS)-induced acute visceral pain, specific alteration of rhythm in temperature (SART) stress-induced chronic visceral pain, and normal defecation. Results T-3047928 (1-10 mg/kg, p.o.) demonstrated a dose-dependent inhibition on oCRH-induced ACTH secretion. In disease models, T-3047928 suppressed fecal pellet output induced by CFS and improved both acute and chronic visceral hypersensitivity induced by RS and SART stress, respectively. Alosetron was also efficacious in stress-induced defecation and visceral pain models at 1 and 10 mg/kg, respectively. Alosetron, however, also suppressed normal defecation at lower those. On the other hand, T-3047928 did not change normal defecation even at higher dose than those in disease models. Conclusion T-3047928 is an orally active CRF1 antagonist that demonstrated potent inhibitory effects in stress-associated IBS models with no effect on normal defecation. Therefore, it is suggested that T-3047928 may have a potency as a novel option for IBS-D therapy with minimal constipation risk.

Proteasomes of pathogenic microbes have become attractive targets for anti-infectives. Co-evolving with its human host, Mycobacterium tuberculosis (Mtb) has developed mechanisms to resist host-imposed nitrosative and oxidative stresses. Genetic deletion or pharmacological inhibition of the Mtb proteasome (Mtb20S) renders non-replicating Mtb susceptible to reactive nitrogen species in vitro and unable to survive in the lungs of mice, validating the Mtb proteasome as a promising target for anti-Mtb agents. Using a structure-guided and flow chemistry-enabled study of structure-activity relationships, we developed phenylimidazole-based peptidomimetics that are highly potent for Mtb20S. X-ray structures of selected compounds with Mtb20S shed light on their selectivity for mycobacterial over human proteasomes.

[Image: see text] The integrin αVβ3 receptor has been implicated in several important diseases, but no antagonists are approved for human therapy. One possible limitation of current small-molecule antagonists is their ability to induce a major conformational change in the receptor that induces it to adopt a high-affinity ligand-binding state. In response, we used structural inferences from a pure peptide antagonist to design the small-molecule pure antagonists TDI-4161 and TDI-3761. Both compounds inhibit αVβ3-mediated cell adhesion to αVβ3 ligands, but do not induce the conformational change as judged by antibody binding, electron microscopy, X-ray crystallography, and receptor priming studies. Both compounds demonstrated the favorable property of inhibiting bone resorption in vitro, supporting potential value in treating osteoporosis. Neither, however, had the unfavorable property of the αVβ3 antagonist cilengitide of paradoxically enhancing aortic sprout angiogenesis at concentrations below its IC(50), which correlates with cilengitide’s enhancement of tumor growth in vivo.

Compound 1 exhibits potent binding inhibition activity against a corticotropin-releasing factor 1 (CRF1) receptor (IC50=9.5nM) and in vitro antagonistic activity (IC50=88nM) but is rapidly metabolized by human hepatic microsomes (182μL/min/mg). Here we identified metabolically stable compounds with potent CRF binding inhibitory activity. Structure-activity relationship (SAR) studies considering in vitro metabolic stability revealed that 4-chloro-2-(2,4-dichloro-6-methylphenoxy)-1-methyl-7-(pentan-3-yl)-1H-benzimidazole 24d was more stable in human microsomes (87μL/min/mg) than compound 1. Compound 24d demonstrated potent CRF binding inhibitory activity (IC50=4.1nM), in vitro antagonistic activity (IC50=44nM), and slow dissociation from the CRF1 receptor. Orally administered compound 24d (6-24μmol/kg) showed ex vivo CRF1 receptor binding in the rat pituitary, olfactory bulb, and frontal cortex and suppressed stress-induced adrenocorticotropic hormone (ACTH) secretion. In this report, we discuss SAR studies on the metabolic stability as well as CRF binding inhibitory activity of the benzimidazole series as CRF1 receptor antagonists and the pharmacological profiles of compound 24d.

Age-associated alterations in the mucosal immune system are generally termed mucosal immunosenescence. The major change seen in the aged mucosa is a failure to elicit an antigen-specific secretory IgA (SIgA) antibody response, which is a central player for host defense from various pathogens at mucosal surfaces. In this regard, it would be a first priority to compensate for mucosal dysregulation in the elderly in order to maintain their health in aging. We have successfully established antigen-specific SIgA antibody responses in aged (2 years old) mice, which provide protective immunity from Streptococcus pneumoniae and influenza virus infections, by using a new adjuvant system consisting of a plasmid encoding Flt3 ligand (pFL) and CpG ODN. In order to explore possible use of current mucosal vaccine strategies for the elderly, we have adoptively transferred adipose tissue-derived mesenchymal stem cells (AMSCs) to aged mice prior to mucosal vaccination. This immune therapy successfully resulted in protective antigen-specific antibody responses in the intestinal mucosa of aged mice that were comparable to those seen in young adult mice. In this regard, we postulate that adoptively transferred AMSCs could augment dendritic cell functions in aged mice. The potential cellular and molecular mechanisms whereby AMSCs restore mucosal immunity in immunosenescence are discussed in this short review. A stem cell transfer system could be an attractive and effective immunologic intervention strategy to reverse mucosal immunosenescence.

Significance Protozoal proteasome is a validated target for antimalarial drug development, but species selectivity of reported inhibitors is suboptimal. Here we identify inhibitors with improved selectivity for malaria proteasome β5 subunit over each active subunit of human proteasomes. These compounds kill the parasite in each stage of its life cycle. They interact synergistically with a β2 inhibitor and with artemisinin. Resistance to the β5 inhibitor arose through a point mutation in the nonproteolytic β6 subunit. The same mutation made the mutant strain more sensitive to a β2 inhibitor and less fit to withstand irradiation. These findings reveal complex interplay among proteasome subunits and introduce the prospect that combined inhibition of β2 and β5 subunits can afford synergy and thwart resistance., We describe noncovalent, reversible asparagine ethylenediamine (AsnEDA) inhibitors of the Plasmodium falciparum proteasome (Pf20S) β5 subunit that spare all active subunits of human constitutive and immuno-proteasomes. The compounds are active against erythrocytic, sexual, and liver-stage parasites, against parasites resistant to current antimalarials, and against P. falciparum strains from patients in Africa. The β5 inhibitors synergize with a β2 inhibitor in vitro and in mice and with artemisinin. P. falciparum selected for resistance to an AsnEDA β5 inhibitor surprisingly harbored a point mutation in the noncatalytic β6 subunit. The β6 mutant was resistant to the species-selective Pf20S β5 inhibitor but remained sensitive to the species-nonselective β5 inhibitors bortezomib and carfilzomib. Moreover, resistance to the Pf20S β5 inhibitor was accompanied by increased sensitivity to a Pf20S β2 inhibitor. Finally, the β5 inhibitor-resistant mutant had a fitness cost that was exacerbated by irradiation. Thus, used in combination, multistage-active inhibitors of the Pf20S β5 and β2 subunits afford synergistic antimalarial activity with a potential to delay the emergence of resistance to artemisinins and each other.

Benzazole derivatives with a flexible aryl group bonded through a one-atom linker as a new scaffold for a corticotropin-releasing factor 1 (CRF1) receptor antagonist were designed, synthesized, and evaluated. We expected that structural diversity could be expanded beyond that of reported CRF1 receptor antagonists. In a structure-activity relationship study, 4-chloro-N(2)-(4-chloro-2-methoxy-6-methylphenyl)-1-methyl-N(7),N(7)-dipropyl-1H-benzimidazole-2,7-diamine 29g had the most potent binding activity against a human CRF1 receptor and the antagonistic activity (IC50 = 9.5 and 88 nM, respectively) without concerns regarding cytotoxicity at 30 μM. Potent CRF1 receptor-binding activity in brain in an ex vivo test and suppression of stress-induced activation of the hypothalamus-pituitary-adrenocortical (HPA) axis were also observed at 138 μmol/kg of compound 29g after oral administration in mice. Thus, the newly designed benzimidazole 29g showed in vivo CRF1 receptor antagonistic activity and good brain penetration, indicating that it is a promising lead for CRF1 receptor antagonist drug discovery research.

Streptococcus pneumoniae (the pneumococcus) causes a major upper respiratory tract infection often leading to severe illness and death in the elderly. Thus, it is important to induce safe and effective mucosal immunity against this pathogen in order to prevent pnuemocaccal infection. However, this is a very difficult task to elicit protective mucosal IgA antibody responses in older individuals. A combind nasal adjuvant consisting of a plasmid encoding the Flt3 ligand cDNA (pFL) and CpG oligonucleotide (CpG ODN) successfully enhanced S. pneumoniae-specific mucosal immunity in aged mice. In particular, a pneumococcal surface protein A-based nasal vaccine given with pFL and CpG ODN induced complete protection from S. pneumoniae infection. These results show that nasal delivery of a combined DNA adjuvant offers an attractive potential for protection against the pneumococcus in the elderly.

A new class of corticotropin releasing factor 1 (CRF1) receptor antagonists characterized by a tricyclic core ring was designed and synthesized. Novel tricyclic derivatives 2a–e were designed as CRF1 receptor antagonists based on conformation analysis of our original 2-anilinobenzimidazole CRF1 receptor antagonist. The synthesized tricyclic derivatives 2a–e showed CRF1 receptor binding activity with IC50 values of less than 400 nM, and the 1,2,3,4-tetrahydropyrimido-[1,2-a]benzimidazole derivative 2e was selected as a lead compound with potent in vitro CRF1 receptor binding activity (IC50 = 7.1 nM). To optimize the pharmacokinetic profiles of lead compound 2e, we explored suitable substituents on the 1-position and 6-position, leading to the identification of compound 42c-R, which exhibited potent CRF1 receptor binding activity (IC50 = 58 nM) with good oral bioavailability (F = 68% in rats). Compound 42c-R exhibited dose-dependent inhibition of [125I]-CRF binding in the frontal cortex (5 and 10 mg/kg, p.o.) as well as suppression of locomotor activation induced by intracerebroventricular administration of CRF in rats (10 mg/kg, p.o.). These results suggest that compound 42c-R successfully binds CRF1 receptors in the brain and exhibits the potential to be further examined for clinical studies.

Cytoplasmic dyneins are motor proteins in the AAA+ superfamily that transport cellular cargos toward microtubule minus-ends. Recently, ciliobrevins were reported as selective cell-permeable inhibitors of cytoplasmic dyneins. As is often true for first-in-class inhibitors, the use of ciliobrevins has in part been limited by low potency. Moreover, suboptimal chemical properties, such as the potential to isomerize, have hindered efforts to improve ciliobrevins. Here, we characterized the structure of ciliobrevins and designed conformationally constrained isosteres. These studies identified dynapyrazoles, inhibitors more potent than ciliobrevins. At single-digit micromolar concentrations dynapyrazoles block intraflagellar transport in the cilium and lysosome motility in the cytoplasm, processes that depend on cytoplasmic dyneins. Further, we find that while ciliobrevins inhibit both dynein's microtubule-stimulated and basal ATPase activity, dynapyrazoles strongly block only microtubule-stimulated activity. Together, our studies suggest that chemical-structure-based analyses can lead to inhibitors with improved properties and distinct modes of inhibition. DOI: http://dx.doi.org/10.7554/eLife.25174.001

G protein-coupled receptor 52 (GPR52) agonists are expected to improve the symptoms of psychiatric disorders. During exploration for a novel class of GPR52 agonists with good pharmacokinetic profiles, we synthesized 4-(3-(3-fluoro-5-(trifluoromethyl)benzyl)-5-methyl-1H-1,2,4-triazol-1-yl)-2-methylbenzamide (4u; half maximal effective concentration (EC50)=75nM, maximal response (Emax)=122%) starting from a high-throughput screening hit 3 (EC50=470nM, Emax=56%). The structural features of a reported GPR52 agonist were applied to 3, led to design 4-azolylbenzamides as novel GPR52 agonists. A structure-activity relationship study of 4-azolylbenzamide resulted in the design of the 1,2,4-triazole derivative 4u, which demonstrated excellent bioavailability in rats (F=53.8%). Oral administration of 4u (10mg/kg) significantly suppressed methamphetamine-induced hyperlocomotion in mice. Thus, 4u is a promising lead compound for drug discovery research of GPR52 agonists.

The aim of this study was to investigate peripheral and central roles of corticotropin-releasing factor (CRF) in endocrinological and behavioral changes. Plasma adrenocorticotropin (ACTH) concentration was measured as an activity of hypothalamic-pituitary-adrenal (HPA) axis. As behavioral changes, locomotion and anxiety behavior were measured after CRF challenge intravenously (i.v.) for the peripheral administration or intracerebroventricularly (i.c.v.) for the central administration. Plasma ACTH concentration was significantly increased by both administration routes of CRF; however, hyperlocomotion and anxiety behavior were induced only by the i.c.v. administration. In the drug discovery of CRF

Compound 1 exhibits potent binding inhibition activity against a corticotropin-releasing factor 1 (CRF

A promising lead compound 1 of a benzimidazole series has been identified as a corticotropin-releasing factor 1 (CRF1) receptor antagonist. In this study, we focused on replacement of a 7-alkylamino group of 1, predicted to occupy a large lipophilic pocket of a CRF1 receptor, with an aryl group. During the course of this examination, we established new synthetic approaches to 2,7-diarylaminobenzimidazoles. The novel synthesis of 7-arylaminobenzimidazoles culminated in the identification of compounds exhibiting inhibitory activities comparable to the alkyl analog 1. A representative compound, p-methoxyanilino analog 16g, showed potent CRF binding inhibitory activity against a human CRF1 receptor and human CRF1 receptor antagonistic activity (IC50=27nM, 56nM, respectively). This compound exhibited ex vivo (125)I-Tyr(0) ((125)I-CRF) binding inhibitory activity in mouse frontal cortex, olfactory bulb, and pituitary gland at 20mg/kg after oral administration. In this report, we discuss the structure-activity-relationship of these 7-arylamino-1H-benzimidazoles and their synthetic method.

The majority of patients with Alzheimer disease (AD) suffer from impaired cerebral circulation. Accumulating evidence suggests that fibrinogen, the main protein component of blood clots, plays an important role in this circulatory dysfunction in AD. Fibrinogen interacts with β-amyloid (Aβ), forming plasmin-resistant abnormal blood clots, and increased fibrin deposition is found in the brains of AD patients and mouse models. In this study, we investigated the biochemical and structural details of the Aβ-fibrinogen interaction. We identified the central region of Aβ42 as the most critical region for the interaction, which can be inhibited by specific antibodies against the central region of Aβ and by naturally occurring p3 peptides, Aβ17-40 and Aβ17-42. X-ray crystallographic analysis revealed that Aβ42 binding to fragment D of fibrinogen induced a structural change in the C-terminal region of the fibrinogen β-chain (β384-393). Furthermore, we identified an additional Aβ-binding site within the αC region of fibrinogen. Aβ binding to this αC region blocked plasmin-mediated fibrin cleavage at this site, resulting in the generation of increased levels of a plasmin-resistant fibrin degradation fragment. Overall, our study elucidates the Aβ-fibrinogen interaction and clarifies the mechanism by which Aβ-fibrinogen binding delays fibrinolysis by plasmin. These results may facilitate the development of effective therapeutics against the Aβ-fibrinogen interaction to treat cerebrovascular abnormalities in AD.

It has been shown that adipose-derived mesenchymal stem cells (AMSCs) can differentiate into adipocytes, chondrocytes and osteoblasts. Several clinical trials have shown the ability of AMSCs to regenerate these differentiated cell types. Age-associated dysregulation of the gastrointestinal (GI) immune system has been well documented. Our previous studies showed that impaired mucosal immunity in the GI tract occurs earlier during agingthan is seen in the systemic compartment. In this study, we examined the potential of AMSCs to restore the GI mucosal immune system in aged mice. Aged (>18 mo old) mice were adoptively transferred with AMSCs. Two weeks later, mice were orally immunized with ovalbumin (OVA) plus cholera toxin (CT) three times at weekly intervals. Seven days after the final immunization, when fecal extract samples and plasma were subjected to OVA- and CT-B-specific ELISA, elevated levels of mucosal secretory IgA (SIgA) and plasma IgG antibody (Ab) responses were noted in aged mouse recipients. Similar results were also seen aged mice which received AMSCs at one year of age. When cytokine production was examined, OVA-stimulated Peyer's patch CD4+ T cells produced increased levels of IL-4. Further, CD4+ T cells from the lamina propria revealed elevated levels of IL-4 and IFN-γ production. In contrast, aged mice without AMSC transfer showed essentially no OVA- or CT-B-specific mucosal SIgA or plasma IgG Ab or cytokine responses. Of importance, fecal extracts from AMSC transferred aged mice showed neutralization activity to CT intoxication. These results suggest that AMSCs can restore impaired mucosal immunity in the GI tract of aged mice.

We report herein the discovery and optimization of 3-amino-1,5-dihydro-4H-pyrazolopyridin-4-one TYK2 inhibitors. High-throughput screening against TYK2 and JAK1-3 provided aminoindazole derivative 1 as a hit compound. Scaffold hopping of the aminoindazole core led to the discovery of 3-amino-1,5-dihydro-4H-pyrazolopyridin-4-one derivative 3 as a novel chemotype of TYK2 inhibitors. Interestingly, initial SAR study suggested that this scaffold could have a vertically flipped binding mode, which prompted us to introduce a substituent at the 7-position as a moiety directed toward the solvent-exposed region. Introduction of a 1-methyl-3-pyrazolyl moiety at the 7-position resulted in a dramatic increase in TYK2 inhibitory activity, and further optimization led to the discovery of 20. Compound 20 inhibited IL-23-induced IL-22 production in a rat PD assay, as well as inhibited IL-23 signaling in human PBMC. Furthermore, 20 showed selectivity for IL-23 signaling inhibition against GM-CSF, demonstrating the unique cytokine selectivity of the novel TYK2 inhibitor.

症例は27歳, 女性. IgA腎症による慢性腎不全のため'94年12月に血液透析に導入となった. '94年7月から特発性血小板減少性紫斑病のためプレドニゾロン (PSL) の大量経口投与を受けていたが, 同薬減量中に血小板減少に伴う紫斑が出現し, 当科へ入院した. PSLを増量 (60mg/日) したが効果は持続せず, 6週間目より漸減を開始した. 9月中旬より38℃台の発熱, 乾性咳が出現した. 各種培養は陰性で抗生剤も無効であった. 胸写にて明らかな異常を認めなかったが, 軽度の低酸素血症があり, 胸部CTにて全肺野に不均一な間質性陰影を認めた. 気管支鏡下に採取されたBAL液よりPneumocystis cariniiの胞体を検出し, カリニ肺炎と診断した. 9月26日よりpentamidine 300mg/日の吸入とsulfamethoxazole-trimethoprim合剤2400mg/日の経口投与を開始し, さらに真菌とcytomegalovirusの混合感染を想定してfluconazole 50mg/日とganciclovir 100mg/隔日の経静脈投与を行った. カリニ肺炎の経過は良好であったが, 10月5日に突然強い嘔吐と両下肢の振戦および夜間不穏が出現した. ganciclovir中止後これらの症状は消失したためganciclovir脳症と考えた. 同薬の血中濃度を測定したが非透析日の半減期は著しく延長し, 投与53時間後も2.91μg/lの高濃度を維持していた. 文献的にganciclovirは血液脳関門を通過することが知られており, 同薬の脳内蓄積が脳症の原因と考えられた. カリニ肺炎は進行が早く致死的疾患であり, しばしば真菌やcytomegalovirusの混合感染が見られる. 本症例ではカリニ肺炎を早期診断し, 治癒しえた意義は大変大きいと思われる. しかし, ganciclovir脳症をきたし, 透析患者におけるganciclovirの投与方法の確立が望まれる.

We describe the system for screening the effective antifolate antimalarials that uses the recombinant Plasmodium falciparum DHFR domain of the bifunctional DHFR-TS expressed in Escherichia coli, and were designed with amino acid alterations found in the DHFR genes of the antifolate resistant strains. The validity of the screen was verified by the subsequent examination of several substituted pyrrolo[2,3-d]pyrimidines for their antimalarial activity. Among the 120 chemical derivatives, 5 compounds were identified by their preferential inhibition of the drug sensitive pfDHFR to that of the mammalian isoenzyme. As compared to the sensitive enzyme, the decrease in response of the cycolguanil-resistant and pyrimethamine-resistant enzymes to the selected compounds were relatively moderate. This gave folds decrease in sensitivity of 0.8-7.5 and 3.6-29, respectively, while those for cycloguanil and pyrimethamine were 400 and 308. The compounds inhibited the growth of drug-sensitive cultured P. falciparum with 50% effective concentrations of the ranged 0.17-30 nM. As contrasted with the sensitive strain, the fold decrease in sensitivity of the resistant parasites were 0.9-2 and 15-50 in the case of the test compounds, while those for cycloguanil and pyrimethamine were 690 and 20,500. Moreover, the most selective pyrrolo-pyrimidine (P-1) showed in vivo activity against P. berghei in mice.

Novel pyrrolo[2, 3-d]pyrimidine antifolates (1a, b and 2a, b) with a nitrogen atom in the bridge chain between the 2, 4-diaminopyrrolo[2, 3-d]pyrimidine and phenylene rings were designed and efficiently synthesized. These compounds exhibited more potent inhibitory activities than methotrexate (MTX) against the proliferation of human epidermoid carcinoma KB cells and human non-small cell lung carcinoma A549 cells despite their modest dihydrofolate reductase (DHFR)-inhibitory potency.

A girl with intractable absence seizures and facial myoclonia at age 7 years was eventually diagnosed with hyperinsulinism-hyperammonemia syndrome because of hypoglycemia, hyperinsulinism, hyperammonemia, and the results of an oral l-leucine loading test. Her seizures occurred even during periods of relatively normal blood glucose levels, and were completely suppressed by diazoxide treatment only. Her diagnosis of hyperinsulinism-hyperammonemia syndrome was confirmed by a loss of sensitivity of glutamate dehydrogenase for guanosine 5'-triphosphate. Genetic studies identified the I444M mutation in the GLUD1 gene, which encodes glutamate dehydrogenase. This case illustrates the complex relationship between seizures and hypoglycemia in hyperinsulinism-hyperammonemia syndrome that can create diagnostic difficulties. The possibility of hyperinsulinism-hyperammonemia syndrome should be considered in patients with refractory absence seizures with myoclonia.

Congenital hyperinsulinism and hyperammonemia (CHH) is caused by gain of function of glutamate dehydrogenase (GDH). The genetic abnormalities are known to be located in three specific regions on the GDH protein. We describe here three different missense mutations identified in five new Japanese patients with CHH. And to study the genotype-phenotype correlations in patients with GLUD1 mutations, we analyzed previously reported Japanese cases.An Epstein-Barr virus-transformed lymphoblastoid cell line was established from the 5 patients and control subjects, and was used for enzymatic and molecular analyses.All patients developed seizures with loss of consciousness associated with hypoglycemia and had persistent hyperammonemia. All patients had similar basal GDH activity of lymphoblasts and insensitivity to GTP inhibition. Genetic studies identified heterozygous I444M mutation in Patient 11, S217C mutation in Patient 1, and H262Y mutation in Patients 2, 3, and 4. Patients 3 and 4 were child and father, respectively. COS cell expression study confirmed that I444M and H262Y mutations were disease-causing genes.We identified three mutations (I444M, H262Y, and S217C), and the former is a newly described mutation. A summary of 17 reported Japanese patients (10 boys and 7 girls) with GDH mutations showed 8 patients had mutation at the site of the GTP-binding region, 2 at the site of the antenna-like structure, and 7 at the site of the hinge region. Analysis of the reported cases showed no clear association between clinical phenotype and mutation sites. However, G446D mutation seems to be associated with serious abnormalities.

Melanin-concentrating hormone receptor 1 (MCHR1) antagonists have been studied as potential agents for the treatment of obesity. Initial structure–activity relationship studies of in-house hit compound 1a and subsequent optimization studies resulted in the identification of tetrahydroisoquinoline derivative 23, 1-(2-acetyl-1,2,3,4-tetrahydroisoquinolin-7-yl)-4-[4-(4-chlorophenyl)piperidin-1-yl]butan-1-one, as a potent hMCHR1 antagonist. A homology model of hMCHR1 suggests that these compounds interact with Asn294 and Asp123 in the binding site of hMCHR1 to enhance binding affinity. Oral administration of compound 23 dose-dependently reduced food intake in diet-induced obesity (DIO)-F344 rats.

Phenylketonuria (PKU) is a heterogeneous metabolic disorder caused by a deficiency in hepatic phenylalanine hydroxylase (PAH). On the basis of phenotype/genotype correlations, determination of phenylketonuric genotype is important for classification of the clinical phenotype and treatment of PKU, including tetrahydrobiopterin therapy. We characterized the genotypes of 203 Japanese patients with PKU and hyperphenylalaninemia using the following systems: (1) denaturing high-performance liquid chromatography with a GC-clamped primer; (2) direct sequencing; and, (3) multiplex ligation-dependent probe amplification. Of 406 mutant alleles, 390 (96%) were genotyped; 65 mutations were identified, including 22 new mutations. R413P, R241C, IVS4-1g>a, R111X and R243Q were prevalent mutations. Mutations prevalent in the Japanese cohort are also common in Korean and Northern Chinese populations, suggesting same origin. The spectrum of prevalent mutations was not significantly different among six Japanese districts, indicating that Japan comprises a relatively homogeneous ethnic group. We classified the mutations by clinical phenotypes and in vivo PAH activity and estimated the mutations with potential tetrahydrobiopterin (BH(4)) responsiveness. The frequency of BH(4) responsiveness based on the genotype was 29.1% in Japanese PKU patients. A catalog of PKU genotypes would be useful for predicting clinical phenotype, deciding on the subsequent treatment of PKU including BH(4) therapy, and genetic counseling in East Asia.

Novel pyrrolo[2, 3-d]pyrimidine antifolates (1a, b and 2a, b) with a nitrogen atom in the bridge chain between the 2, 4-diaminopyrrolo[2, 3-d]pyrimidine and phenylene rings were designed and efficiently synthesized. These compounds exhibited more potent inhibitory activities than methotrexate (MTX) against the proliferation of human epidermoid carcinoma KB cells and human non-small cell lung carcinoma A549 cells despite their modest dihydrofolate reductase (DHFR)-inhibitory potency.

A series of novel pyrrolo[2,3-d]pyrimidine derivatives was designed and synthesized as thymidylate synthase (TS) inhibitors. Molecular design was performed on the human TS complex model built on the basis of the reported structure of TS-deoxyuridinemonophosphate (dUMP)-CB3717 ternary complex. From a docking study, we expected that a one-carbon bridge between pyrrolo[2,3-d]pyrimidine and an aromatic ring was suitable. Moreover, we found that the bridge carbon could be replaced with an alkyl group to fill out the unoccupied space. Based on this design, we synthesized five pyrrolo[2,3-d]pyrimidine derivatives with one-carbon bridge and evaluated their TS inhibitory activities. All synthesized compounds inhibited TS more potently than compound 2 (LY231514), and the C8-ethyl analogue (7) showed a remarkable inhibitory activity against TS (IC50=0.017 μM).

血液透析療法 (HD) を必要とした多発性骨髄腫 (multiple myeloma: MM) 9例について, その臨床経過と検査所見を透析離脱例と非離脱例に分けて解析し, 腎機能回復に及ぼす因子について検討した. 9例中5例は化学療法などの積極的治療により腎機能が回復し, HDを離脱した (離脱群), 残りの4例はHDの継続を必要とした (非離脱群). HD導入時の離脱群の平均年齢は非離脱群に比し低い傾向にあったが, MMそのもののstageには差がなかった. また全例でBence Jones蛋白 (BJP) が陽性であったが, その尿中排泄量と腎不全の程度の間に相関はなく, 腎機能回復にも関係なかった. 離脱群のMMの型は1例を除き全例light chain (LC) 型であったのに対し, 非離脱群ではLC型を認めなかった. 腎不全の誘因としてはBJP, 脱水, 高カルシウム血症, 高尿酸血症が両群に共通して認められたが, HD導入時の検査所見は両群間にいずれも差を認めなかった. MM関連症状出現よりHD導入までの期間は, 離脱群は1例を除いて全例1か月以内で, 非離脱群より明らかに短かった. また, 離脱群は1例以外は急性腎不全による発症で, その原因にLCが考えられた. 一方, 非離脱群は慢性腎不全からの増悪例が多く, アミロイドーシスによる心不全が, 腎機能の回復を妨げた-因であった. 以上より, MM関連症状出現よりHD導入までの期間, 腎不全の発症形式, アミロイドーシスの合併などの所見は腎機能の回復に重大な影響を及ぼす因子と考えられた.

A novel series of 4-phenylisoquinolones were synthesized and evaluated as c-Jun N-terminal kinase (JNK) inhibitors. Initial modification at the 2- and 3-positions of the isoquinolone ring of hit compound 4, identified from high-throughput screening, led to the lead compound 6b. The optimization was carried out using a JNK1-binding model of 6b and several compounds exhibited potent JNK inhibition. Among them, 11g significantly inhibited cardiac hypertrophy in rat pressure-overload models without affecting blood pressure and the concept of JNK inhibitors as novel therapeutic agents for heart failure was confirmed.