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There remain no approved therapies for rare but devastating neuronopathic glyocosphingolipid storage diseases, such as Sandhoff, Tay-Sachs, and Gaucher disease type 3. We previously reported initial optimization of the scaffold of eliglustat, an approved therapy for the peripheral symptoms of Gaucher disease type 1, to afford 2, which effected modest reductions in brain glucosylceramide (GlcCer) in normal mice at 60 mg/kg. The relatively poor pharmacokinetic properties and high Pgp-mediated efflux of 2 prompted further optimization of the scaffold. With a general objective of reducing topological polar surface area, and guided by multiple metabolite identification studies, we were successful at identifying 17 (CCG-222628), which achieves remarkably greater brain exposure in mice than 2. After demonstrating an over 60-fold improvement in potency over 2 at reducing brain GlcCer in normal mice, we compared 17 with Sanofi clinical candidate venglustat (Genz-682452) in the CBE mouse model of Gaucher disease type 3. At doses of 10 mg/kg, 17 and venglustat effected comparable reductions in both brain GlcCer and glucosylsphingosine. Importantly, 17 achieved these equivalent pharmacodynamic effects at significantly lower brain exposure than venglustat.

As part of a program toward making analogues of amlexanox (1), currently under clinical investigation for the treatment of type 2 diabetes and obesity, we have synthesized derivative 5 in which deuterium has been introduced into two sites of metabolism on the C-7 isopropyl function of amlexanox. The synthesis of 5 was completed in an efficient three-step process utilizing reduction of key olefin 7b to 8 by Wilkinson’s catalyst to provide specific incorporation of di-deuterium across the double bond. Compound 5 displayed nearly equivalent potency to amlexanox (IC(50) 1.1 μM vs 0.6 μM, respectively) against recombinant human TBK1. When incubated with human, rat, and mouse liver microsomes, amlexanox (1) and d(2)-amlexanox (5) were stable (t(1/2) > 60 min) with 1 showing marginally greater stability relative to 5 except for rat liver microsomes. These data show that incorporating deuterium into two sites of metabolism does not majorly suppress Cyp-mediated metabolism relative to amlexanox.

Item does not contain fulltext BACKGROUND: Congenital anomalies of the kidney and urinary tract (CAKUT) are the most prevalent cause of kidney disease in the first three decades of life. Previous gene panel studies showed monogenic causation in up to 12% of patients with CAKUT. METHODS: We applied whole-exome sequencing to analyze the genotypes of individuals from 232 families with CAKUT, evaluating for mutations in single genes known to cause human CAKUT and genes known to cause CAKUT in mice. In consanguineous or multiplex families, we additionally performed a search for novel monogenic causes of CAKUT. RESULTS: In 29 families (13%), we detected a causative mutation in a known gene for isolated or syndromic CAKUT that sufficiently explained the patient's CAKUT phenotype. In three families (1%), we detected a mutation in a gene reported to cause a phenocopy of CAKUT. In 15 of 155 families with isolated CAKUT, we detected deleterious mutations in syndromic CAKUT genes. Our additional search for novel monogenic causes of CAKUT in consanguineous and multiplex families revealed a potential single, novel monogenic CAKUT gene in 19 of 232 families (8%). CONCLUSIONS: We identified monogenic mutations in a known human CAKUT gene or CAKUT phenocopy gene as the cause of disease in 14% of the CAKUT families in this study. Whole-exome sequencing provides an etiologic diagnosis in a high fraction of patients with CAKUT and will provide a new basis for the mechanistic understanding of CAKUT. 01 september 2018

Blockade of the renin–angiotensin system (RAS) has been shown to alleviate inflammatory processes in the gastrointestinal tract. The aim of this study was to determine if blockade of the RAS would be effective in an immunologically relevant colitis model, and to compare outcome with an acute colitis model. A losartan analog, CCG-203025 (C23H26ClN3O5S) containing a highly polar sulfonic acid moiety that we expected would allow localized mucosal antagonism with minimal systemic absorption was selected as an angiotensin II type 1a receptor antagonist (AT1aR-A). Two colitis models were studied: (1) Acute colitis was induced in 8- to 10-week-old C57BL/6J mice by 2.5 % dextran sodium sulfate (DSS, in drinking water) for 7 days. (2) IL10-/-colitis Piroxicam (200 ppm) was administered orally in feed to 5-week-old IL-10-/-mice (C57BL/6J background) for 14 days followed by enalaprilat (ACE-I), CCG-203025 or PBS administered transanally for 14 days. In the DSS model, weight loss and histologic score for CCG-203025 were better than with placebo. In the IL10-/-model, ACE-I suppressed histologic damage better than CCG-203025. Both ACE-I and CCG-203025 reduced pro-inflammatory cytokines and chemokines. This study demonstrated the therapeutic efficacy of both ACE-I and AT1aR-A for preventing the development of both acute and immunologically relevant colitis.

Studies demonstrate that small molecule targeting of atypical protein kinase C (aPKC) may provide an effective means to control vascular permeability, prevent edema, and reduce inflammation providing novel and important alternatives to anti-VEGF therapies for certain blinding eye diseases. Based on a literature tricyclic thieno[2,3-d]pyrimidine lead (1), an ATP-competitive inhibitor of the aPKC iota (ι) and aPKC zeta (ζ) isoforms, we have synthesized a small series of compounds in 1-2 steps from a readily available chloro intermediate. A single pyridine congener was also made using 2D NMR to assign regiochemistry. Within the parent pyrimidine series, a range of potencies was observed against aPKCζ whereas the pyridine congener was inactive. Selected compounds were also tested for their effect toward VEGF-induced permeability in BREC cells. The most potent of these (7l) was further assayed against the aPKCι isoform and showed a favorable selectivity profile against a panel of 31 kinases, including kinases from the AGC superfamily, with a focus on PKC isoforms and kinases previously shown to affect permeability. Further testing of 7l in a luciferase assay in HEK293 cells showed an ability to prevent TNF-α induced NFκB activation while not having any effect on cell survival. Intravitreal administration of 7l to the eye yielded a complete reduction in permeability in a test to determine whether the compound could block VEGF- and TNFα-induced permeability across the retinal vasculature in a rat model. The compound in mice displayed good microsomal stability and in plasma moderate exposure (AUC and Cmax), low clearance, a long half-life and high oral bioavailability. With IV dosing, higher levels were observed in the brain and eye relative to plasma, with highest levels in the eye by either IV or PO dosing. With a slow oral absorption profile, 7l accumulates in the eye to maintain a high concentration after dosing with higher levels than in plasma. Compound 7l may represent a class of aPKC inhibitors for further investigation.

We recently reported the development of a novel inhibitor of Rho-mediated gene transcription (1, CCG-203971) that is efficacious in multiple animal models of acute fibrosis, including scleroderma, when given intraperitoneally. The modest in vivo potency and poor pharmacokinetics (PK) of this lead, however, make it unsuitable for long term efficacy studies. We therefore undertook a systematic medicinal chemistry effort to improve both the metabolic stability and the solubility of 1, resulting in the identification of two analogs achieving over 10-fold increases in plasma exposures in mice. We subsequently showed that one of these analogs (8f, CCG-232601) could inhibit the development of bleomycin-induced dermal fibrosis in mice when administered orally at 50 mg/kg, an effect that was comparable to what we had observed earlier with 1 at a 4-fold higher IP dose.

To combat emerging coronaviruses, developing safe and efficient platforms to evaluate viral protease activities and the efficacy of protease inhibitors is a high priority. Here, we exploit a biosafety level 2 (BSL-2) chimeric Sindbis virus system to evaluate protease activities and the efficacy of inhibitors directed against the papain-like protease (PLpro) of severe acute respiratory syndrome coronavirus (SARS-CoV), a biosafety level 3 (BSL-3) pathogen. We engineered Sindbis virus to coexpress PLpro and a substrate, murine interferon-stimulated gene 15 (ISG15), and found that PLpro mediates removal of ISG15 (deISGylation) from cellular proteins. Mutation of the catalytic cysteine residue of PLpro or addition of a PLpro inhibitor blocked deISGylation in virus-infected cells. Thus, deISGylation is a marker of PLpro activity. Infection of alpha/beta interferon receptor knockout (IFNAR −/− ) mice with these chimeric viruses revealed that PLpro deISGylation activity removed ISG15-mediated protection during viral infection. Importantly, administration of a PLpro inhibitor protected these mice from lethal infection, demonstrating the efficacy of a coronavirus protease inhibitor in a mouse model. However, this PLpro inhibitor was not sufficient to protect the mice from lethal infection with SARS-CoV MA15, suggesting that further optimization of the delivery and stability of PLpro inhibitors is needed. We extended the chimeric-virus platform to evaluate the papain-like protease/deISGylating activity of Middle East respiratory syndrome coronavirus (MERS-CoV) to provide a small-animal model to evaluate PLpro inhibitors of this recently emerged pathogen. This platform has the potential to be universally adaptable to other viral and cellular enzymes that have deISGylating activities. IMPORTANCE Evaluating viral protease inhibitors in a small-animal model is a critical step in the path toward antiviral drug development. We modified a biosafety level 2 chimeric virus system to facilitate evaluation of inhibitors directed against highly pathogenic coronaviruses. We used this system to demonstrate the in vivo efficacy of an inhibitor of the papain-like protease of severe acute respiratory syndrome coronavirus. Furthermore, we demonstrate that the chimeric-virus system can be adapted to study the proteases of emerging human pathogens, such as Middle East respiratory syndrome coronavirus. This system provides an important tool to rapidly assess the efficacy of protease inhibitors targeting existing and emerging human pathogens, as well as other enzymes capable of removing ISG15 from cellular proteins.

Structure-guided design was used to generate a series of noncovalent inhibitors with nanomolar potency against the papain-like protease (PLpro) from the SARS coronavirus (CoV). A number of inhibitors exhibit antiviral activity against SARS-CoV infected Vero E6 cells and broadened specificity toward the homologous PLP2 enzyme from the human coronavirus NL63. Selectivity and cytotoxicity studies established a more than 100-fold preference for the coronaviral enzyme over homologous human deubiquitinating enzymes (DUBs), and no significant cytotoxicity in Vero E6 and HEK293 cell lines is observed. X-ray structural analyses of inhibitor-bound crystal structures revealed subtle differences between binding modes of the initial benzodioxolane lead (15g) and the most potent analogues 3k and 3j, featuring a monofluoro substitution at para and meta positions of the benzyl ring, respectively. Finally, the less lipophilic bis(amide) 3e and methoxypyridine 5c exhibit significantly improved metabolic stability and are viable candidates for advancing to in vivo studies.

Resistance to antibiotics is an increasingly dire threat to human health that warrants the development of new modes of treating infection. We recently identified 1 (CCG-2979) as an inhibitor of the expression of streptokinase, a critical virulence factor in Group A Streptococcus that endows blood-borne bacteria with fibrinolytic capabilities. In this report, we describe the synthesis and biological evaluation of a series of novel 5,6-dihydrobenzo[h]quinazolin-4(3H)-one analogs of 1 undertaken with the goal of improving the modest potency of the lead. In addition to achieving an over 35-fold increase in potency, we identified structural modifications that improve the solubility and metabolic stability of the scaffold. The efficacy of two new compounds 12c (CCG-203592) and 12k (CCG-205363) against biofilm formation in Staphylococcus aureus represents a promising additional mode of action for this novel class of compounds.

In heart failure, the β-adrenergic receptors (βARs) become desensitized and uncoupled from heterotrimeric G proteins. This process is initiated by G protein-coupled receptor kinases (GRKs), some of which are upregulated in the failing heart, making them desirable therapeutic targets. The selective serotonin reuptake inhibitor, paroxetine, was previously identified as a GRK2 inhibitor. Utilizing a structure-based drug design approach, we modified paroxetine to generate a small compound library. Included in this series is a highly potent and selective GRK2 inhibitor, 14as, with an IC50 of 30 nM against GRK2 and greater than 230-fold selectivity over other GRKs and kinases. Furthermore, 14as showed a 100-fold improvement in cardiomyocyte contractility assays over paroxetine and a plasma concentration higher than its IC50 for over 7 h. Three of these inhibitors, including 14as, were additionally crystallized in complex with GRK2 to give insights into the structural determinants of potency and selectivity of t...

Synthesis inhibition is the basis for the treatment of type 1 Gaucher disease by the glucosylceramide synthase (GCS) inhibitor eliglustat tartrate. However, the extended use of eliglustat and related compounds for the treatment of glycosphingolipid storage diseases with CNS manifestations is limited by the lack of brain penetration of this drug. Property modeling around the D-threo-1-phenyl-2-decanoylamino-3-morpholino-propanol (PDMP) pharmacophore was employed in a search for compounds of comparable activity against the GCS but lacking P-glycoprotein (MDR1) recognition. Modifications of the carboxamide N-acyl group were made to lower total polar surface area and rotatable bond number. Compounds were screened for inhibition of GCS in crude enzyme and whole cell assays and for MDR1 substrate recognition. One analog, 2-(2,3-dihydro-1H-inden-2-yl)-N-((1R,2R)-1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1-hydroxy-3-(pyrrolidin-1-yl)propan-2-yl)acetamide (CCG-203586), was identified that inhibited GCS at low nanomolar concentrations with little to no apparent recognition by MDR1. Intraperitoneal administration of this compound to mice for 3 days resulted in a significant dose dependent decrease in brain glucosylceramide content, an effect not seen in mice dosed in parallel with eliglustat tartrate.

Sandhoff disease is an incurable neurodegenerative disorder caused by mutations in the lysosomal hydrolase β-hexosaminidase. Deficiency in this enzyme leads to excessive accumulation of ganglioside GM2 and its asialo derivative, GA2, in brain and visceral tissues. Small molecule inhibitors of ceramide-specific glucosyltransferase, the first committed step in ganglioside biosynthesis, reduce storage of GM2 and GA2. Limited brain access or adverse effects have hampered the therapeutic efficacy of the clinically approved substrate reduction molecules, eliglustat tartrate and the imino sugar NB-DNJ (Miglustat). The novel eliglustat tartrate analog, 2-(2,3-dihydro-1H-inden-2-yl)-N-((1R,2R)-1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1-hydroxy-3-(pyrrolidin-1-yl)propan-2-yl)acetamide (EtDO-PIP2, CCG-203586 or “3h”), was recently reported to reduce glucosylceramide in murine brain. Here we assessed the therapeutic efficacy of 3h in juvenile Sandhoff (Hexb −/−) mice. Sandhoff mice received intraperitoneal injections of phosphate buffered saline (PBS) or 3h (60 mg/kg/day) from postnatal day 9 (p-9) to postnatal day 15 (p-15). Brain weight and brain water content was similar in 3h and PBS-treated mice. 3h significantly reduced total ganglioside sialic acid, GM2, and GA2 content in cerebrum, cerebellum and liver of Sandhoff mice. Data from the liver showed that 3h reduced the key upstream ganglioside precursor (glucosylceramide), providing direct evidence for an on target mechanism of action. No significant differences were seen in the distribution of cholesterol or of neutral and acidic phospholipids. These data suggest that 3h can be an effective alternative to existing substrate reduction molecules for ganglioside storage diseases.

To discover dual inhibitors of 5-lipoxygenase (LO) and cyclooxygenase (CO) with improved pharmacokinetic properties, we have designed and synthesized series of 1,2,4-triazole, 1,3,4-oxadiazole, and 1,3,4-thiadiazole di-tert-butylphenol derivatives which exhibit a wide range of log P (2.3 to > 4) and pKa (5.5-12) values. From this work 5-[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]-1,3,4-thiadiazole-2(3H)- thione, choline salt (12a, CI-986) was found to be a potent inhibitor of 5-LO (IC50 = 2.8 microM) and CO (IC50 = 0.8 microM), orally active in rat models of inflammation and nonulcerogenic.

The aim of this work was to study in mammals the ability of high pressures to reverse the anesthesia produced by a wide range of general anesthetics. Dose-response curves were obtained using mice at pressures ranging from 1 to 125 atm for five agents, namely alpha-chloralose, ethylcarbamate, phenobarbital and, for comparison, nitrogen and argon. The increase of ED50 was found to be a linear function of pressure in each case, but the proportionate increases in ED50 with pressure were greater for the three non-inhalation agents than for the two gases. Thus, the ratio of ED50 at 100 atm to that at 1 atm was 1.74 for alpha-chloralose, 1.68 for ethylcarbamate, and 1.54 for phenobarbital. On the other hand, the corresponding ratios for argon and nitrogen were only 1.36 and 1.34. The potencies of three short-acting agents (trichloroethanol, ketamine, and alphadione) were shown to increase with decreasing pressure, although ED50 values could not be obtained. It is concluded that pressure reverses the actions of a wide variety of anesthetics in mice. The results of this study are not inconsistent with either the fluidized lipid membrane or the critical volume hypotheses of anesthetic action.