Development of CVN424: A Selective and Novel GPR6 Inverse Agonist Effective in Models of Parkinson Disease.

GPR6 is an orphan G-protein-coupled receptor that has enriched expression in the striatopallidal, indirect pathway and medium spiny neurons of the striatum. This pathway is greatly impacted by the loss of the nigro-striatal dopaminergic neurons in Parkinson disease, and modulating this neurocircuitry can be therapeutically beneficial. In this study, we describe the in vitro and in vivo pharmacological characterization of (R)-1-(2-(4-(2,4-difluorophenoxy)piperidin-1-yl)-3-((tetrahydrofuran-3-yl)amino)-7,8-dihydropyrido[3,4-b]pyrazin-6(5H)-yl)ethan-1-one (CVN424), a highly potent and selective small-molecule inverse agonist for GPR6 that is currently undergoing clinical evaluation. CVN424 is brain-penetrant and shows dose-dependent receptor occupancy that attained brain 50% of receptor occupancy at plasma concentrations of 6.0 and 7.4 ng/ml in mice and rats, respectively. Oral administration of CVN424 dose-dependently increases locomotor activity and reverses haloperidol-induced catalepsy. Furthermore, CVN424 restored mobility in bilateral 6-hydroxydopamine lesion model of Parkinson disease. The presence and localization of GPR6 in medium spiny neurons of striatum postmortem samples from both nondemented control and patients with Parkinson disease were confirmed at the level of both RNA (using Nuclear Enriched Transcript Sort sequencing) and protein. This body of work demonstrates that CVN424 is a potent, orally active, and brain-penetrant GPR6 inverse agonist that is effective in preclinical models and is a potential therapeutic for improving motor function in patients with Parkinson disease. SIGNIFICANCE STATEMENT: CVN424 represents a nondopaminergic novel drug for potential use in patients with Parkinson disease.

Using Target Engagement Biomarkers to Predict Clinical Efficacy of MetAP2 Inhibitors.

Target-engagement pharmacodynamic (PD) biomarkers are valuable tools in the prioritization of drug candidates, especially for novel, first-in-class mechanisms whose robustness to alter disease outcome is unknown. Methionine aminopeptidase 2 (MetAP2) is a cytosolic metalloenzyme that cleaves the N-terminal methionine from nascent proteins. Inhibition of MetAP2 leads to weight loss in obese rodents, dogs and humans. However, there is a need to develop efficacious compounds that specifically inhibit MetAP2 with an improved safety profile. The objective of this study was to identify a PD biomarker for selecting potent, efficacious compounds and for predicting clinical efficacy that would result from inhibition of MetAP2. Here we report the use of NMet14-3-3γ for this purpose. Treatment of primary human cells with MetAP2 inhibitors resulted in an approx. 10-fold increase in NMet14-3-3γ levels. Furthermore, treatment of diet-induced obese mice with these compounds reduced body weight (approx. 20%) and increased NMet14-3-3γ (approx. 15-fold) in adipose tissues. The effects on target engagement and body weight increased over time and were dependent on dose and administration frequency of compound. The relationship between compound concentration in plasma, NMet14-3-3γ in tissue, and reduction of body weight in obese mice was used to generate a pharmacokinetic-pharmacodynamic-efficacy model for predicting efficacy of MetAP2 inhibitors in mice. We also developed a model for predicting weight loss in humans using a target engagement PD assay that measures inhibitor-bound MetAP2 in blood. In summary, MetAP2 target engagement biomarkers can be used to select efficacious compounds and predict weight loss in humans. SIGNIFICANCE STATEMENT: The application of target engagement pharmacodynamic biomarkers during drug development provides a means to determine the dose required to fully engage the intended target and an approach to connect the drug target to physiological effects. This work exemplifies the process of using target engagement biomarkers during preclinical research to select new drug candidates and predict clinical efficacy. We determine concentration of MetAP2 antiobesity compounds needed to produce pharmacological activity in primary human cells and in target tissues from an appropriate animal model and establish key relationships between pharmacokinetics, pharmacodynamics, and efficacy, including the duration of effects after drug administration. The biomarkers described here can aid decision-making in early clinical trials of MetAP2 inhibitors for the treatment of obesity.

Effective targeting of Hedgehog signaling in a medulloblastoma model with PF-5274857, a potent and selective Smoothened antagonist that penetrates the blood-brain barrier.

Inhibition of the Smoothened (Smo) represents a promising therapeutic strategy for treating malignant tumors that are dependent on the Hedgehog (Hh) signaling pathway. PF-5274857 is a novel Smo antagonist that specifically binds to Smo with a K(i) of 4.6 ± 1.1 nmol/L and completely blocks the transcriptional activity of the downstream gene Gli1 with an IC(50) of 2.7 ± 1.4 nmol/L in cells. This Smo antagonist showed robust antitumor activity in a mouse model of medulloblastoma with an in vivo IC(50) of 8.9 ± 2.6 nmol/L. The downregulation of Gli1 is closely linked to the tumor growth inhibition in patched(+/-) medulloblastoma mice. Mathematical analysis of the relationship between the drug's pharmacokinetics and Gli1 pharmacodynamics in patched(+/-) medulloblastoma tumor models yielded similar tumor and skin Gli1 IC(50) values, suggesting that skin can be used as a surrogate tissue for the measurement of tumor Gli1 levels. In addition, PF-5274857 was found to effectively penetrate the blood-brain barrier and inhibit Smo activity in the brain of primary medulloblastoma mice, resulting in improved animal survival rates. The brain permeability of PF-5274857 was also confirmed and quantified in nontumor-bearing preclinical species with an intact blood-brain barrier. PF-5274857 was orally available and metabolically stable in vivo. These findings suggest that PF-5274857 is a potentially attractive clinical candidate for the treatment of tumor types including brain tumors and brain metastasis driven by an activated Hh pathway.

Effects of p38 MAPK inhibition on early stages of diabetic retinopathy and sensory nerve function.

Purpose. p38 mitogen-activated protein kinase (MAPK) is known to play a regulatory role in inflammatory processes in disease. Inflammation has been linked also to the development of diabetic retinopathy in rodents. This study was conducted to evaluate the effect of a p38 MAPK inhibitor on the development of early stages of diabetic retinopathy in rats. Methods. Streptozotocin-diabetic rats were assigned to two groups-treated with the p38 MAPK inhibitor PHA666859 (Pfizer, New York, NY) and untreated-and compared with age-matched nondiabetic control animals. Results. At 2 months of diabetes, insulin-deficient diabetic control rats exhibited significant increases in retinal superoxide, nitric oxide (NO), cyclooxygenase (COX)-2, and leukostasis within retinal microvessels. All these abnormalities were significantly inhibited by the p38 MAPK inhibitor (25 mg/kgBW/d). At 10 months of diabetes, significant increases in the number of degenerate (acellular) capillaries and pericyte ghosts were measured in control diabetic rats versus those in nondiabetic control animals, and pharmacologic inhibition of p38 MAPK significantly inhibited all these abnormalities (all P < 0.05). This therapy also had beneficial effects outside the eye in diabetes, as evidenced by the inhibition of a diabetes-induced hypersensitivity of peripheral nerves to light touch (tactile allodynia). Conclusions. p38 MAPK plays an important role in diabetes-induced inflammation in the retina, and inhibition of p38 MAPK offers a novel therapeutic approach to inhibiting the development of early stages of diabetic retinopathy and other complications of diabetes.

Discovery of tricyclic 5,6-dihydro-1H-pyridin-2-ones as novel, potent, and orally bioavailable inhibitors of HCV NS5B polymerase.

A novel series of non-nucleoside small molecules containing a tricyclic dihydropyridinone structural motif was identified as potent HCV NS5B polymerase inhibitors. Driven by structure-based design and building on our previous efforts in related series of molecules, we undertook extensive SAR studies, in which we identified a number of metabolically stable and very potent compounds in genotype 1a and 1b replicon assays. This work culminated in the discovery of several inhibitors, which combined potent in vitro antiviral activity against both 1a and 1b genotypes, metabolic stability, good oral bioavailability, and high C(12) (PO)/EC(50) ratios.

5,5'- and 6,6'-dialkyl-5,6-dihydro-1H-pyridin-2-ones as potent inhibitors of HCV NS5B polymerase.

The discovery of 5,5'- and 6,6'-dialkyl-5,6-dihydro-1H-pyridin-2-ones as potent inhibitors of the HCV RNA-dependent RNA polymerase (NS5B) is described. Several of these agents also display potent antiviral activity in cell culture experiments (EC50 <0.10 microM). In vitro DMPK data for selected compounds as well as crystal structures of representative inhibitors complexed with the NS5B protein are also disclosed.

5,6-Dihydro-1H-pyridin-2-ones as potent inhibitors of HCV NS5B polymerase.

5,6-Dihydro-1H-pyridin-2-one analogs were discovered as a novel class of inhibitors of genotype 1 HCV NS5B polymerase. Among these, compound 4ad displayed potent inhibitory activities in biochemical and replicon assays (IC(50) (1b)<10nM; IC(50) (1a)<25nM, EC(50) (1b)=16nM), good in vitro DMPK properties, as well as moderate oral bioavailability in monkeys (F=24%).

Hexahydro-pyrrolo- and hexahydro-1H-pyrido[1,2-b]pyridazin-2-ones as potent inhibitors of HCV NS5B polymerase.

Hexahydro-pyrrolo- and hexahydro-1H-pyrido[1,2-b]pyridazin-2-one analogs were discovered as a novel class of inhibitors of genotype 1 HCV NS5B polymerase. Among these, compound 4c displayed potent inhibitory activities in biochemical and replicon assays (IC(50) (1b) <10 nM; EC(50) (1b)=34 nM) as well as good stability towards human liver microsomes (HLM t(1/2) =59 min).

4-(1,1-Dioxo-1,4-dihydro-1lambda6-benzo[1,4]thiazin-3-yl)-5-hydroxy-2H-pyridazin-3-ones as potent inhibitors of HCV NS5B polymerase.

4-(1,1-Dioxo-1,4-dihydro-1lambda(6)-benzo[1,4]thiazin-3-yl)-5-hydroxy-2H-pyridazin-3-one analogs were discovered as a novel class of inhibitors of HCV NS5B polymerase. Structure-based design led to the identification of compound 3a that displayed potent inhibitory activities in biochemical and replicon assays (1b IC(50)<10 nM; 1b EC(50)=1.1 nM) as well as good stability toward human liver microsomes (HLM t(1/2)>60 min).

Structure-based design, synthesis, and biological evaluation of 1,1-dioxoisothiazole and benzo[b]thiophene-1,1-dioxide derivatives as novel inhibitors of hepatitis C virus NS5B polymerase.

A novel series of HCV NS5B polymerase inhibitors comprising 1,1-dioxoisothiazoles and benzo[b]thiophene-1,1-dioxides were designed, synthesized, and evaluated. SAR studies guided by structure-based design led to the identification of a number of potent NS5B inhibitors with nanomolar IC(50) values. The most potent compound exhibited IC(50) less than 10nM against the genotype 1b HCV polymerase and EC(50) of 70 nM against a genotype 1b replicon in cell culture. The DMPK properties of selected compounds were also evaluated.

Pyrrolo[1,2-b]pyridazin-2-ones as potent inhibitors of HCV NS5B polymerase.

Pyrrolo[1,2-b]pyridazin-2-one analogs were discovered as a novel class of inhibitors of genotype 1 HCV NS5B polymerase. Structure-based design led to the discovery of compound 3 k, which displayed potent inhibitory activities in biochemical and replicon assays (IC(50) (1b)<10nM; EC(50) (1b)=12 nM) as well as good stability towards human liver microsomes (HLM t(1/2)>60 min).

Novel HCV NS5B polymerase inhibitors derived from 4-(1',1'-dioxo-1',4'-dihydro-1'lambda(6)-benzo[1',2',4']thiadiazin-3'-yl)-5-hydroxy-2H-pyridazin-3-ones. Part 3: Further optimization of the 2-, 6-, and 7'-substituents and initial pharmacokinetic assessments.

5-Hydroxy-3(2H)-pyridazinone derivatives were investigated as inhibitors of genotype 1 HCV NS5B polymerase. Lead optimization led to the discovery of compound 3a, which displayed potent inhibitory activities in biochemical and replicon assays [IC(50) (1b)<10nM; IC(50) (1a)=22 nM; EC(50) (1b)=5nM], good stability toward human liver microsomes (HLM t(1/2)>60 min), and high ratios of liver to plasma concentrations 12h after a single oral administration to rats.

Novel HCV NS5B polymerase inhibitors derived from 4-(1',1'-dioxo-1',4'-dihydro-1'lambda6-benzo[1',2',4']thiadiazin-3'-yl)-5-hydroxy-2H-pyridazin-3-ones: Part 4. Optimization of DMPK properties.

5-Hydroxy-3(2H)-pyridazinone derivatives were investigated as potent inhibitors of genotype 1 HCV NS5B polymerase focusing on the optimization of their drug metabolism and pharmacokinetics (DMPK) profiles. This investigation led to the discovery of potent inhibitors with improved DMPK properties.

Discovery of ANA975: an oral prodrug of the TLR-7 agonist isatoribine.

ANA975, a 5-amino-3-beta -D-ribofuranosyl-3H-thiazolo[4,5-d]pyrimidin-2-one derivative, was synthesized in the search of an oral prodrug of isatoribine, a small molecule toll-like receptor 7 (TLR-7) agonist. Several strategies were studied to enable the kilogram-scale synthesis of ANA975. Three general total syntheses are described. In the phase I clinical study of ANA975 against hepatitis C virus (HCV), conversion to isatoribine in plasma was rapid and effective, delivering levels of isatoribine that have been shown to be clinically relevant.