Greet Vanhoof

An antisense-based functional genomics approach for identification of genes critical for growth of Candida albicans.

Wenonah Vercoutere, Stephen Winters-Hilt, Hugh Olsen, David Deamer, David Haussler, and Mark Akeson Nat. Biotechnol. 19, 248–252 (2001). The URL given for the DNA mfold server in Table 1 (p. 249 ) and in the text (p. 251) is incorrect. The correct URL is http://bioinfo.math.rpi.edu/~mfold/dna/form1.cgiConverting the complete genome sequence of Candida albicans into meaningful biological information will require comprehensive screens for identifying functional classes of genes. Most systems described so far are not applicable to C. albicans because of its difficulty with mating, its diploid nature, and the lack of functional random insertional mutagenesis methods. We examined artificial gene suppression as a means to identify gene products critical for growth of this pathogen; these represent new antifungal drug targets. To achieve gene suppression we combined antisense RNA inhibition and promoter interference. After cloning antisense complementary DNA (cDNA) fragments under control of an inducible GAL1 promoter, we transferred the resulting libraries to C. albicans. Over 2,000 transformant colonies were screened for a promoter-induced diminished-growth phenotype. After recovery of the plasmids, sequence determination of their inserts revealed the messenger RNA (mRNA) they inhibited or the gene they disrupted. Eighty-six genes critical for growth were identified, 45 with unknown function. When used in high-throughput screening for antifungals, the crippled C. albicans strains generated in this study showed enhanced sensitivity to specific drugs.

Synthesis, In Vivo Occupancy, and Radiolabeling of Potent Phosphodiesterase Subtype-10 Inhibitors as Candidates for Positron Emission Tomography Imaging

We have recently reported the phosphodiesterase 10A (PDE10A) inhibitor 2-[4-[1-(2-[(18)F]fluoroethyl)-4-pyridin-4-yl-1H-pyrazol-3-yl]-phenoxymethyl]-quinoline ([(18)F]1a) as a promising candidate for in vivo imaging using positron emission tomography (PET). We now describe the synthesis and biological evaluation of a series of related pyridinyl analogues that exhibit high potency and selectivity as PDE10A inhibitors. The most interesting compounds were injected in rats to measure their levels of PDE10A occupancy through an in vivo occupancy assay. The 3,5-dimethylpyridine derivative 3 and the 5-methoxypyridine derivative 4 showed a comparable level of occupancy to that of 1a. Because these derivatives showed lower in vitro activity and are slightly less lipophilic than 1a, we hypothesized that they could behave as better PET imaging ligands. Compounds [(18)F]3, [(18)F]4, and [(11)C]4 were radiosynthesized and subjected to biodistribution studies in rats for a preliminary evaluation as candidate PET radioligands for in vivo imaging of PDE10A in the brain.

Discovery of a potent, selective, and orally active phosphodiesterase 10A inhibitor for the potential treatment of schizophrenia.

We report the discovery of a series of imidazo[1,2-a]pyrazine derivatives as novel inhibitors of phosphodiesterase 10A (PDE10A). In a high-throughput screening campaign we identified the imidazopyrazine derivative 1, a PDE10A inhibitor with limited selectivity versus the other phosphodiesterases (PDEs). Subsequent investigation of 1 and replacement of the trimethoxyphenyl group by a (methoxyethyl)pyrazole moiety maintained PDE10A inhibition but enhanced selectivity against the other PDEs. Systematic examination and analysis of structure-activity and structure-property relationships resulted in the discovery of 2, an in vitro potent and selective inhibitor of PDE10A with high striatal occupancy of PDE10A, promising in vivo efficacy in different rodent behavioral models of schizophrenia, and a good pharmacokinetic profile in rats.

Discovery of a new series of [1,2,4]triazolo[4,3-a]quinoxalines as dual phosphodiesterase 2/phosphodiesterase 10 (PDE2/PDE10) inhibitors.

The synthesis, preliminary evaluation and structure-activity relationship (SAR) of a series of 1-aryl-4-methyl[1,2,4]triazolo[4,3-a]quinoxalines as dual phosphodiesterase 2/phosphodiesterase 10 (PDE2/PDE10) inhibitors are described. From this investigation compound 31 was identified, showing good combined potency, acceptable brain uptake and high selectivity for both PDE2 and PDE10 enzymes. Compound 31 was subjected to a microdosing experiment in rats, showing preferential distribution in brain areas where both PDE2 and PDE10 are highly expressed. These promising results may drive the further development of highly potent combined PDE2/PDE10 inhibitors, or even of selective inhibitors of PDE2 and/or PDE10.

Pyrido[4,3-e][1,2,4]triazolo[4,3-a]pyrazines as Selective, Brain Penetrant Phosphodiesterase 2 (PDE2) Inhibitors.

A novel series of pyrido[4,3-e][1,2,4]triazolo[4,3-a]pyrazines is reported as potent PDE2/PDE10 inhibitors with drug-like properties. Selectivity for PDE2 was obtained by introducing a linear, lipophilic moiety on the meta-position of the phenyl ring pending from the triazole. The SAR and protein flexibility were explored with free energy perturbation calculations. Rat pharmacokinetic data and in vivo receptor occupancy data are given for two representative compounds 6 and 12.

Structure-Based Design of a Potent, Selective, and Brain Penetrating Pde2 Inhibitor with Demonstrated Target Engagement.

Structure-guided design led to the identification of the novel, potent, and selective phosphodiesterase 2 (PDE2) inhibitor 12. Compound 12 demonstrated a >210-fold selectivity versus PDE10 and PDE11 and was inactive against all other PDE family members up to 10 μM. In vivo evaluation of 12 provided evidence that it is able to engage the target and to increase cGMP levels in relevant brain regions. Hence, 12 is a valuable tool compound for the better understanding of the role of PDE2 in cognitive impairment and other central nervous system related disorders.

Patterns of brain glucose metabolism induced by phosphodiesterase-10A inhibitors in the mouse: a potential translational biomarker

Phosphodiesterase 10A (PDE10A) inhibitors have recently been proposed as a new therapy for schizophrenia. The aim of this study was to enhance our understanding of the role of PDE10A inhibitors and potentially identify a clinically useful mechanistic/functional biomarker by using 2-deoxyglucose (2-DG) autoradiography. PDE10A inhibitors papaverine (10 and 40 mg/kg), 6,7-dimethoxy-4-[(3R)-3-(2-quinoxalinyloxy)-1-pyrrolidinyl]quinazoline (PQ-10), (0.16–10 mg/kg), and 2-[{4-(1-methyl-4-pyridin-4-yl-1H-pyrazol-3-yl)phenoxy}methyl]quinoline (MP-10) (0.16–40 mg/kg) induced region-specific hypermetabolism in the globus pallidus and lateral habenula of C57BL/6 mice. Studies with MP-10 revealed a dose-dependent relative increase in globus pallidus activation, whereas a bell-shaped curve was observed for the lateral habenula. Although the relative increase in 2-DG uptake in the lateral habenula was also characteristic of the D2 antagonist haloperidol (0.01–0.63 mg/kg), relative 2-DG changes were absent in the globus pallidus. This observation probably is explained by the interaction of PDE10A inhibitors with the D1 direct pathway as suggested by experiments in combination with the D1 agonist (±)-6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide (SKF-82958) (0.16 mg/kg). The absence of an effect of MP-10 (2.5 mg/kg) on relative glucose metabolism in the globus pallidus and lateral habenula of PDE10A knockout mice confirmed the specificity of the signal induced by PDE10A inhibitors. These studies substantiate the regulatory role of PDE10A in the basal ganglia circuit and as such support the potential of PDE10A inhibitors for treating psychiatric disorders. Moreover, we could differentiate PDE10A inhibitors from haloperidol based on specific patterns of hypermetabolism probably caused by its combined action at both direct and indirect dopaminergic pathways. Finally, these specific changes in brain glucose metabolism may act as a translational biomarker for target engagement in future clinical studies.

Pharmacology of JNJ-42314415, a Centrally Active Phosphodiesterase 10A (PDE10A) Inhibitor: A Comparison of PDE10A Inhibitors with D2 Receptor Blockers as Potential Antipsychotic Drugs

The new phosphodiesterase 10A inhibitor (PDE10AI) JNJ-42314415 [3-[6-(2-methoxyethyl)pyridin-3-yl]-2-methyl-8-morpholin-4-ylimidazo[1,2-a]pyrazine] was compared with three reference PDE10AIs and eight dopamine 2 (D2) receptor blockers. Despite displaying relatively low PDE10A activity in vitro, JNJ-42314415 was found to be a relatively potent and specific PDE10AI in vivo. The compound was devoid of effects on prolactin release and of receptor interactions associated with other commonly observed adverse effects of available antipsychotics. Similar to D2 receptor blockers, the tested PDE10AIs antagonized stimulant-induced behavior and inhibited conditioned avoidance behavior; these effects were observed at doses close to the ED50 for striatal PDE10A occupancy. Relative to the ED50 for inhibition of apomorphine-induced stereotypy, PDE10AIs blocked conditioned avoidance behavior and behaviors induced by nondopaminergic stimulants (phencyclidine, scopolamine) more efficiently than did D2 receptor blockers; however, they blocked behaviors induced by dopaminergic stimulants (apomorphine, d-amphetamine) less efficiently. PDE10AIs also induced less pronounced catalepsy than D2 receptor blockers. The effects of PDE10A inhibition against dopaminergic stimulants and on catalepsy were potentiated by the D1 antagonist SCH-23390 (8-chloro-3-methyl-5-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepin-7-ol), suggesting that enhancement of D1 receptor-mediated neurotransmission contributes to the behavioral profile of PDE10AIs. By reducing dopamine D2 and concomitantly potentiating dopamine D1 receptor-mediated neurotransmission, PDE10AIs may show antipsychotic activity with an improved side-effect profile relative to D2 receptor blockers. However, the clinical implications of this dual mechanism must be further explored.

PDE10A inhibitors stimulate or suppress motor behavior dependent on the relative activation state of the direct and indirect striatal output pathways

The enzyme phosphodiesterase 10A (PDE10A) regulates the activity of striatal, medium spiny neurons (MSNs), which are divided into a behaviorally stimulating, Gs‐coupled D1 receptor‐expressing “direct” pathway and a behaviorally suppressant, Gi‐coupled D2 receptor‐expressing “indirect” pathway. Activating both pathways, PDE10A inhibitors (PDE10AIs) combine functional characteristics of D2 antagonists and D1 agonists. While the effects of PDE10AIs on spontaneous and stimulated behavior have been extensively reported, the present study investigates their effects on suppressed behavior under various conditions of reduced dopaminergic neurotransmission: blockade of D1 receptors with SCH‐23390, blockade of D2 receptors with haloperidol, or depletion of dopamine with RO‐4‐1284 or reserpine. In rats, PDE10AIs displayed relatively low cataleptic activity per se. After blocking D1 receptors, however, they induced pronounced catalepsy at low doses close to those required for inhibition of apomorphine‐induced behavior; slightly higher doses resulted in behavioral stimulant effects, counteracting the catalepsy. PDE10AIs also counteracted catalepsy and related behaviors induced by D2 receptor blockade or dopamine depletion; catalepsy was replaced by behavioral stimulant effects under the latter but not the former condition. Similar interactions were observed at the level of locomotion in mice. At doses close to those inhibiting d‐amphetamine‐induced hyperlocomotion, PDE10AIs reversed hypolocomotion induced by D1 receptor blockade or dopamine depletion but not hypolocomotion induced by D2 receptor blockade. It is concluded that PDE10AIs stimulate or inhibit motor behavior dependent on the relative activation state of the direct and indirect striatal output pathways.

Genetic deletion of PDE10A selectively impairs incentive salience attribution and decreases medium spiny neuron excitability.

The striatum is the main input structure to the basal ganglia and consists mainly out of medium spiny neurons. The numerous spines on their dendrites render them capable of integrating cortical glutamatergic inputs with a motivational dopaminergic signal that originates in the midbrain. This integrative function is thought to underly attribution of incentive salience, a process that is severely disrupted in schizophrenic patients. Phosphodiesterase 10A (PDE10A) is located mainly to the striatal medium spiny neurons and hydrolyses cAMP and cGMP, key determinants of MSN signaling. We show here that genetic depletion of PDE10A critically mediates attribution of salience to reward-predicting cues, evident in impaired performance in PDE10A knockout mice in an instrumentally conditioned reinforcement task. We furthermore report modest impairment of latent inhibition in PDE10A knockout mice, and unaltered prepulse inhibition. We suggest that the lack of effect on PPI is due to the pre-attentional nature of this task. Finally, we performed whole-cell patch clamp recordings and confirm suggested changes in intrinsic membrane excitability. A decrease in spontaneous firing in striatal medium spiny neurons was found. These data show that PDE10A plays a pivotal role in striatal signaling and striatum-mediated salience attribution.