High-throughput Screening Hit Research Articles

Discovery of Imidazo[1,2-a]pyrazines and Pyrazolo[1,5-c]pyrimidines as TARP γ-8 Selective AMPAR Negative Modulators.

This report discloses the discovery and characterization of imidazo[1,2-a]pyrazines and pyrazolo[1,5-c]pyrimidines as selective negative modulators of α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors (AMPARs) associated with transmembrane AMPAR regulatory protein γ-8. Imidazopyrazine 5 was initially identified as a promising γ-8 selective high-throughput screening hit, and subsequent structure-activity relationship optimization yielded subnanomolar, brain penetrant leads. Replacement of the imidazopyrazine core with an isosteric pyrazolopyrimidine scaffold improved microsomal stability and efflux liabilities to provide 26, JNJ-61432059. Following oral administration, 26 exhibited time- and dose-dependent AMPAR/γ-8 receptor occupancy in mouse hippocampus, which resulted in robust seizure protection in corneal kindling and pentylenetetrazole (PTZ) anticonvulsant models.

5-Keto-3-cyano-2,4-diaminothiophenes as selective maternal embryonic leucine zipper kinase inhibitors

Maternal embryonic leucine zipper kinase (MELK) is involved in several key cellular processes and displays increased levels of expression in numerous cancer classes (colon, breast, brain, ovary, prostate and lung). Although no selective MELK inhibitors have yet been approved, increasing evidence suggest that inhibition of MELK would constitute a promising approach for cancer therapy. A weak high-throughput screening hit (17, IC50 ≈ 5 μM) with lead-like properties was optimized for MELK inhibition. The early identification of a plausible binding mode by molecular modeling offered guidance in the choice of modifications towards compound 52 which displayed a 98 nM IC50. A good selectivity profile was achieved for a representative member of the series (29) in a 486 protein kinase panel. Future elaboration of 52 has the potential to deliver compounds for further development with chemotherapeutic aims.

A Drug with Lipophilicity-Dependent Potency Can Be Metabolically Stable: Discovery of a Potent and Selective Retinoic Acid Receptor-Related Orphan Receptor C2 (RORC2) Inverse Agonist as an Orally Bioavailable Anti-Inflammatory Agent.

IL-17 drives an amplification mechanism in inflammatory diseases such as psoriasis, psoriatic arthritis, and ankylosing spondylitis. The production of IL-17 depends on the activity of RORC2 in immune cells, which suggests that RORC2 inverse agonists are capable of anti-inflammatory therapy by reducing IL-17 levels. However, oral delivery of inverse agonists remains a challenge since the binding pocket of RORC2 prefers to accommodate lipophilic ligands, which tend to have poor metabolic stability. This Viewpoint discusses recent results published in this journal that identified a potent, selective, and orally bioavailable RORC2 inverse agonist as an anti-inflammatory agent, optimized from a high-throughput screening (HTS) hit through a combination of de novo and structure-guided design.

Discovery of Small Molecule Splicing Modulators of Survival Motor Neuron-2 (SMN2) for the Treatment of Spinal Muscular Atrophy (SMA).

Spinal muscular atrophy (SMA), a rare neuromuscular disorder, is the leading genetic cause of death in infants and toddlers. SMA is caused by the deletion or a loss of function mutation of the survival motor neuron 1 (SMN1) gene. In humans, a second closely related gene SMN2 exists; however it codes for a less stable SMN protein. In recent years, significant progress has been made toward disease modifying treatments for SMA by modulating SMN2 pre-mRNA splicing. Herein, we describe the discovery of LMI070/branaplam, a small molecule that stabilizes the interaction between the spliceosome and SMN2 pre-mRNA. Branaplam (1) originated from a high-throughput phenotypic screening hit, pyridazine 2, and evolved via multiparameter lead optimization. In a severe mouse SMA model, branaplam treatment increased full-length SMN RNA and protein levels, and extended survival. Currently, branaplam is in clinical studies for SMA.

6-Benzhydryl-4-amino-quinolin-2-ones as Potent Cannabinoid Type 1 (CB1) Receptor Inverse Agonists and Chemical Modifications for Peripheral Selectivity.

A novel series of 6-benzhydryl-4-amino-quinolin-2-ones was discovered as cannabinoid type 1 receptor (CB1R) inverse agonists based on the high-throughput screening hit, compound 1a. Structure-activity relationships were studied to improve in vitro/in vivo pharmacology and restrict distribution to the peripheral circulation. We adopted several strategies such as increasing topological polar surface area, incorporating discrete polyethylene glycol side chains, and targeting P-glycoprotein (P-gp) to minimize access to the brain. Compound 6a is a P-gp substrate and a potent and highly selective CB1R inverse agonist, demonstrating excellent in vivo metabolic stability and a low brain to plasma ratio. However, brain receptor occupancy studies showed that compound 6a may accumulate in brain with repeat dosing. This was evidenced by compound 6a inhibiting food intake and inducing weight loss in diet-induced obese mice. Thus, a strategy based on P-gp efflux may not be adequate for peripheral restriction of the disclosed quinolinone series.

Optimization and Lead Selection of Benzothiazole Amide Analogs Toward a Novel Antimycobacterial Agent.

Mycobacteria remain an important problem worldwide, especially drug resistant human pathogens. Novel therapeutics are urgently needed to tackle both drug-resistant tuberculosis (TB) and difficult-to-treat infections with nontuberculous mycobacteria (NTM). Benzothiazole adamantyl amide had previously emerged as a high throughput screening hit against M. tuberculosis (Mtb) and was subsequently found to be active against NTM as well. For lead optimization, we applied an iterative process of design, synthesis and screening of several 100 analogs to improve antibacterial potency as well as physicochemical and pharmacological properties to ultimately achieve efficacy. Replacement of the adamantyl group with cyclohexyl derivatives, including bicyclic moieties, resulted in advanced lead compounds that showed excellent potency and a mycobacteria-specific spectrum of activity. MIC values ranged from 0.03 to 0.12 μg/mL against M. abscessus (Mabs) and other rapid- growing NTM, 1–2 μg/mL against M. avium complex (MAC), and 0.12–0.5 μg/mL against Mtb. No pre-existing resistance was found in a collection of n = 54 clinical isolates of rapid-growing NTM. Unlike many antibacterial agents commonly used to treat mycobacterial infections, benzothiazole amides demonstrated bactericidal effects against both Mtb and Mabs. Metabolic labeling provided evidence that the compounds affect the transfer of mycolic acids to their cell envelope acceptors in mycobacteria. Mapping of resistance mutations pointed to the trehalose monomycolate transporter (MmpL3) as the most likely target. In vivo efficacy and tolerability of a benzothiazole amide was demonstrated in a mouse model of chronic NTM lung infection with Mabs. Once daily dosing over 4 weeks by intrapulmonary microspray administration as 5% corn oil/saline emulsion achieved statistically significant CFU reductions compared to vehicle control and non-inferiority compared to azithromycin. The benzothiazole amides hold promise for development of a novel therapeutic agent with broad antimycobacterial activity, though further work is needed to develop drug formulations for direct intrapulmonary delivery via aerosol.

Discovery of Novel Aminotetralines and Aminochromanes as Selective and Competitive Glycine Transporter 1 (GlyT1) Inhibitors.

The glycine transporter 1 (GlyT1) has emerged as a key novel target for the treatment of schizophrenia. Herein, we report the synthesis and biological evaluation of aminotetralines and aminochromanes as novel classes of competitive GlyT1 inhibitors. Starting from a high-throughput screening hit, structure-activity relationship studies led first to the discovery of aminotetralines displaying high GlyT1 potency and selectivity, with favorable pharmacokinetic properties. Systematic investigations of various parameters (e.g., topological polar surface area, number of hydrogen bond donors) guided by ex vivo target occupancy evaluation resulted in lead compounds possessing favorable brain penetration properties as for (7 S,8 R)-27a. Further optimization revealed compounds with reduced efflux liabilities as for aminochromane 51b. In an in vivo efficacy model (7 S,8 R)-27a, dose-dependently reversed L-687,414 induced hyperlocomotion in mice with an ED50 of 0.8 mg/kg. All these results suggest (7 S,8 R)-27a and 51b as new GlyT1 inhibitors worthy of further profiling.

Correcting the F508del-CFTR variant by modulating eukaryotic translation initiation factor 3–mediated translation initiation

Inherited and somatic rare diseases result from >200,000 genetic variants leading to loss- or gain-of-toxic function, often caused by protein misfolding. Many of these misfolded variants fail to properly interact with other proteins. Understanding the link between factors mediating the transcription, translation, and protein folding of these disease-associated variants remains a major challenge in cell biology. Herein, we utilized the cystic fibrosis transmembrane conductance regulator (CFTR) protein as a model and performed a proteomics-based high-throughput screen (HTS) to identify pathways and components affecting the folding and function of the most common cystic fibrosis-associated mutation, the F508del variant of CFTR. Using a shortest-path algorithm we developed, we mapped HTS hits to the CFTR interactome to provide functional context to the targets and identified the eukaryotic translation initiation factor 3a (eIF3a) as a central hub for the biogenesis of CFTR. Of note, siRNA-mediated silencing of eIF3a reduced the polysome-to-monosome ratio in F508del-expressing cells, which, in turn, decreased the translation of CFTR variants, leading to increased CFTR stability, trafficking, and function at the cell surface. This finding suggested that eIF3a is involved in mediating the impact of genetic variations in CFTR on the folding of this protein. We posit that the number of ribosomes on a CFTR mRNA transcript is inversely correlated with the stability of the translated polypeptide. Polysome-based translation challenges the capacity of the proteostasis environment to balance message fidelity with protein folding, leading to disease. We suggest that this deficit can be corrected through control of translation initiation.

TISSUE-SELECTIVE ABCA1 AGONISTS AS POTENTIAL ALZHEIMER'S DISEASE THERAPEUTICS: HIT-TO-LEAD OPTIMIZATION

The apolipoprotein E (APOE) ε4 allele is the strongest known risk factor for sporadic Alzheimer's disease (AD), which accounts for over 95% of all AD cases. ApoE4-containing lipoproteins contain a lower lipid content, which decreases stability and contributes to loss of lipoprotein function. To correct these deficits and restore proper lipoprotein function, we have developed tissue-selective ABCA1 agonists (TSAAgs) that induce central nervous system expression of the cholesterol transport protein ABCA1, thereby increasing lipid content of apoE4-containing lipoproteins, with minimal impact on peripheral lipogenesis. TSAAgs also ameliorate additional aspects of AD, including neuroinflammation and insulin resistance. High-throughput screening (HTS) utilized CCF-STTG1 astrocytoma (primary screen) and HepG2 hepatocellular carcinoma cells (counterscreen) expressing luciferase response elements linked to ABCA1 and SREBP1c promoters, respectively. Novel structural analogs of HTS hits were generated via iterative chemical synthesis. In vitro analysis of analog activity included PCR/immunoblot for both lipid- and insulin-related genes, ELISA for inflammatory markers, and a fluorescent cholesterol efflux assay. Prioritized HTS hits – those demonstrating anti-inflammatory and insulin-sensitizing properties in addition to TSAAg activity – served as scaffolds to generate a library of structural analogs. Evaluation of this analog library in vitro established structure-activity relationships that identified compounds with improved TSAAg activity and guided further structural modification. These results demonstrated a proof-of-concept to develop TSAAgs possessing multifunctional therapeutic potential against Alzheimer's disease. Our study represents a novel strategy to develop small molecule drug candidates that target multiple aspects of AD pathology. Future in vivo experiments are proposed to establish pharmacokinetic profiles, determine magnitude and mechanisms of tissue-selective ABCA1 induction, and monitor alterations in peripheral lipogenesis. Finally, treatments in the EFAD mouse model will assess TSAAg effects on cognitive and pathological deficits. Successful completion of this project could result in establishment of TSAAg compounds as leads for further pharmaceutical development and human clinical testing.

Nuisance Compounds, PAINS Filters, and Dark Chemical Matter in the GSK HTS Collection

High-throughput screening (HTS) hits include compounds with undesirable properties. Many filters have been described to identify such hits. Notably, pan-assay interference compounds (PAINS) has been adopted by the community as the standard term to refer to such filters, and very useful guidelines have been adopted by the American Chemical Society (ACS) and subsequently triggered a healthy scientific debate about the pitfalls of draconian use of filters. Using an inhibitory frequency index, we have analyzed in detail the promiscuity profile of the whole GlaxoSmithKline (GSK) HTS collection comprising more than 2 million unique compounds that have been tested in hundreds of screening assays. We provide a comprehensive analysis of many previously published filters and newly described classes of nuisance structures that may serve as a useful source of empirical information to guide the design or growth of HTS collections and hit triaging strategies.

Abstract 2802: Discovery of the first selective and cell-active eIF4A3 inhibitors

Abstract Background: Eukaryotic initiation factor 4A3 (eIF4A3) is a member of the Asp-Glu-Ala-Asp (DEAD) box RNA helicase family. There are three subtypes of eIF4A, eIF4A1, 2, and 3. eIF4A1 and eIF4A2 are required for translation initiation as their name suggest, however eIF4A3 is functionally distinct and one of the core components of the exon junction complex (EJC). The EJC is known to be involved in a variety of RNA metabolic processes typified by nonsense-mediated RNA decay (NMD), which is the surveillance mechanism that recognizes mRNAs containing premature termination codons to prevent the accumulation of truncated proteins. It is known that eIF4A3 is an ATP-dependent RNA clamp that can serve as a nucleation center to recruit other EJC components, and siRNA-mediated knockdown of eIF4A3 leads to a defect in NMD. In order to investigate the functions of eIF4A3 further and evaluate the therapeutic potential, we conducted a search for molecular probes of eIF4A3. Methods: Using an RNA dependent ATPase assay as a guide, intensive structure activity relationship (SAR) study and chemical optimization of high-throughput screening hit were conducted. Thereafter, NMD inhibitory activity, selectivity, helicase inhibitory activity, and physicochemical properties of optimized compounds were confirmed. Additionally, direct binding of inhibitors to eIF4A3 and their binding mode were analyzed using biophysical methods. Results: Optimized compounds showed high selectivity for eIF4A3 and exhibited significant NMD inhibitory activity in HEK293T cells. The drastic difference in eIF4A3 inhibitory activity between eutomers and distomer revealed the importance of stereochemistry at the 3-position of the piperazine ring for eIF4A3 inhibition. The lack of significant NMD inhibition by distomers suggests that NMD inhibition of eutomers is mediated by eIF4A3 inhibition. A surface plasmon resonance (SPR) biosensing assay and hydrogen/deuterium exchange mass spectrometry (HDX MS) provided strong evidence for the direct binding of the inhibitors to eIF4A3 and implications for their binding site. Conclusion: We discovered the first selective eIF4A3 inhibitors exhibiting cellular NMD inhibitory activity. These novel inhibitors not only represent novel molecular probes for investigation of RNA biology but also could be promising lead compounds for drug discovery research.

Database for High Throughput Screening Hits (dHITS): a simple tool to retrieve gene specific phenotypes from systematic screens done in yeast.

In the last decade several collections of Saccharomyces cerevisiae yeast strains have been created. In these collections every gene is modified in a similar manner such as by a deletion or the addition of a protein tag. Such libraries have enabled a diversity of systematic screens, giving rise to large amounts of information regarding gene functions. However, often papers describing such screens focus on a single gene or a small set of genes and all other loci affecting the phenotype of choice (‘hits’) are only mentioned in tables that are provided as supplementary material and are often hard to retrieve or search. To help unify and make such data accessible, we have created a Database of High Throughput Screening Hits (dHITS). The dHITS database enables information to be obtained about screens in which genes of interest were found as well as the other genes that came up in that screen – all in a readily accessible and downloadable format. The ability to query large lists of genes at the same time provides a platform to easily analyse hits obtained from transcriptional analyses or other screens. We hope that this platform will serve as a tool to facilitate investigation of protein functions to the yeast community.

Discovery of Orally Bioavailable Selective Inhibitors of the Sodium-Phosphate Cotransporter NaPi2a (SLC34A1).

Sodium-phosphate cotransporter 2a, or NaPi2a (SLC34A1), is a solute-carrier (SLC) transporter located in the kidney proximal tubule that reabsorbs glomerular-filtered phosphate. Inhibition of NaPi2a may enhance urinary phosphate excretion and correct maladaptive mineral and hormonal derangements associated with increased cardiovascular risk in chronic kidney disease-mineral and bone disorder (CKD-MBD). To date, only nonselective NaPi inhibitors have been described. Herein, we detail the discovery of the first series of selective NaPi2a inhibitors, resulting from optimization of a high-throughput screening hit. The oral PK profile of inhibitor PF-06869206 (6f) in rodents allows for the exploration of the pharmacology of selective NaPi2a inhibition.

Discovery of an Irreversible and Cell-Active BCL6 Inhibitor Selectively Targeting Cys53 Located at the Protein-Protein Interaction Interface.

B-cell lymphoma 6 (BCL6) is the most frequently involved oncogene in diffuse large B-cell lymphomas (DLBCLs). BCL6 shows potent transcriptional repressor activity through interactions with its corepressors, such as BCL6 corepressor (BCOR). The inhibition of the protein-protein interaction (PPI) between BCL6 and its corepressors suppresses the growth of BCL6-dependent DLBCLs, thus making BCL6 an attractive drug target for lymphoma treatment. However, potent small-molecule PPI inhibitor identification remains challenging because of the lack of deep cavities at PPI interfaces. This article reports the discovery of a potent, cell-active small-molecule BCL6 inhibitor, BCL6-i (8), that operates through irreversible inhibition. First, we synthesized irreversible lead compound 4, which targets Cys53 in a cavity on the BCL6-BTB domain dimer by introducing an irreversible warhead to high-throughput screening hit compound 1. Further chemical optimization of 4 based on kinact/KI evaluation produced BCL6-i with a kinact/KI value of 1.9 × 104 M-1 s-1, corresponding to a 670-fold improvement in potency compared to that of 4. By exploiting the property of irreversible inhibition, engagement of BCL6-i to intracellular BCL6 was confirmed. BCL6-i showed intracellular PPI inhibitory activity between BCL6 and its corepressors, thus resulting in BCL6-dependent DLBCL cell growth inhibition. BCL6-i is a cell-active chemical probe with the most potent BCL6 inhibitory activity reported to date. The discovery process of BCL6-i illustrates the utility of irreversible inhibition for identifying potent chemical probes for intractable target proteins.

Role of Chemical Biology in Tuberculosis Drug Discovery and Diagnosis.

The use of chemical techniques to study biological systems (often referred to currently as chemical biology) has become a powerful tool for both drug discovery and the development of novel diagnostic strategies. In tuberculosis, such tools have been applied to identifying drug targets from hit compounds, matching high-throughput screening hits against large numbers of isolated protein targets and identifying classes of enzymes with important functions. Metabolites unique to mycobacteria have provided important starting points for the development of innovative tools. For example, the unique biology of trehalose has provided both novel diagnostic strategies as well as probes of in vivo biological processes that are difficult to study any other way. Other mycobacterial metabolites are potentially valuable starting points and have the potential to illuminate new aspects of mycobacterial pathogenesis.

Discovery of novel, orally bioavailable, antileishmanial compounds using phenotypic screening.

Leishmaniasis is a parasitic infection that afflicts approximately 12 million people worldwide. There are several limitations to the approved drug therapies for leishmaniasis, including moderate to severe toxicity, growing drug resistance, and the need for extended dosing. Moreover, miltefosine is currently the only orally available drug therapy for this infection. We addressed the pressing need for new therapies by pursuing a two-step phenotypic screen to discover novel, potent, and orally bioavailable antileishmanials. First, we conducted a high-throughput screen (HTS) of roughly 600,000 small molecules for growth inhibition against the promastigote form of the parasite life cycle using the nucleic acid binding dye SYBR Green I. This screen identified approximately 2,700 compounds that inhibited growth by over 65% at a single point concentration of 10 μM. We next used this 2700 compound focused library to identify compounds that were highly potent against the disease-causing intra-macrophage amastigote form and exhibited limited toxicity toward the host macrophages. This two-step screening strategy uncovered nine unique chemical scaffolds within our collection, including two previously described antileishmanials. We further profiled two of the novel compounds for in vitro absorption, distribution, metabolism, excretion, and in vivo pharmacokinetics. Both compounds proved orally bioavailable, affording plasma exposures above the half-maximal effective concentration (EC50) concentration for at least 12 hours. Both compounds were efficacious when administered orally in a murine model of cutaneous leishmaniasis. One of the two compounds exerted potent activity against trypanosomes, which are kinetoplastid parasites related to Leishmania species. Therefore, this compound could help control multiple parasitic diseases. The promising pharmacokinetic profile and significant in vivo efficacy observed from our HTS hits highlight the utility of our two-step phenotypic screening strategy and strongly suggest that medicinal chemistry optimization of these newly identified scaffolds will lead to promising candidates for an orally available anti-parasitic drug.

Discovery of orally efficacious RORγt inverse agonists, part 1: Identification of novel phenylglycinamides as lead scaffolds

A series of novel phenylglycinamides as retinoic acid receptor-related orphan receptor-gamma t (RORγt) inverse agonists were discovered through optimization of a high-throughput screen hit 1. (R)-N-(2-((3,5-Difluoro-4-(trimethylsilyl)phenyl) amino)-1-(4-methoxyphenyl)-2-oxoethyl)-3-hydroxy-N-methylisoxazole-5-carboxamide (22) was identified as one of the best of these compounds. It displayed higher subtype selectivity and specificity over other nuclear receptors and demonstrated in vivo potency to suppress the transcriptional activity of RORγt in a mouse PD (pharmacodynamic) model upon oral administration.

Discovery of Small-Molecule Inhibitors of Ubiquitin Specific Protease 7 (USP7) Using Integrated NMR and in Silico Techniques.

USP7 is a deubiquitinase implicated in destabilizing the tumor suppressor p53, and for this reason it has gained increasing attention as a potential oncology target for small molecule inhibitors. Herein we describe the biophysical, biochemical, and computational approaches that led to the identification of 4-(2-aminopyridin-3-yl)phenol compounds described by Kategaya ( Nature 2017 , 550 , 534 - 538 ) as specific inhibitors of USP7. Fragment based lead discovery (FBLD) by NMR combined with virtual screening and re-mining of biochemical high-throughput screening (HTS) hits led to the discovery of a series of ligands that bind in the "palm" region of the catalytic domain of USP7 and inhibit its catalytic activity. These ligands were then optimized by structure-based design to yield cell-active molecules with reasonable physical properties. This discovery process not only involved multiple techniques working in concert but also illustrated a unique way in which hits from orthogonal screening approaches complemented each other for lead identification.

Discovery and Characterization of (R)-6-Neopentyl-2-(pyridin-2-ylmethoxy)-6,7-dihydropyrimido[2,1-c][1,4]oxazin-4(9H)-one (PF-06462894), an Alkyne-Lacking Metabotropic Glutamate Receptor 5 Negative Allosteric Modulator Profiled in both Rat and Nonhuman Primates.

We previously observed a cutaneous type IV immune response in nonhuman primates (NHP) with the mGlu5 negative allosteric modulator (NAM) 7. To determine if this adverse event was chemotype- or mechanism-based, we evaluated a distinct series of mGlu5 NAMs. Increasing the sp3 character of high-throughput screening hit 40 afforded a novel morpholinopyrimidone mGlu5 NAM series. Its prototype, (R)-6-neopentyl-2-(pyridin-2-ylmethoxy)-6,7-dihydropyrimido[2,1-c][1,4]oxazin-4(9H)-one (PF-06462894, 8), possessed favorable properties and a predicted low clinical dose (2 mg twice daily). Compound 8 did not show any evidence of immune activation in a mouse drug allergy model. Additionally, plasma samples from toxicology studies confirmed that 8 did not form any reactive metabolites. However, 8 caused the identical microscopic skin lesions in NHPs found with 7, albeit with lower severity. Holistically, this work supports the hypothesis that this unique toxicity may be mechanism-based although additional work is required to confirm this and determine clinical relevance.

Discovery of an Orally Bioavailable, Brain-Penetrating, in Vivo Active Phosphodiesterase 2A Inhibitor Lead Series for the Treatment of Cognitive Disorders.

Herein, we describe the discovery of a potent, selective, brain-penetrating, in vivo active phosphodiesterase (PDE) 2A inhibitor lead series. To identify high-quality leads suitable for optimization and enable validation of the physiological function of PDE2A in vivo, structural modifications of the high-throughput screening hit 18 were performed. Our lead generation efforts revealed three key potency-enhancing functionalities with minimal increases in molecular weight (MW) and no change in topological polar surface area (TPSA). Combining these structural elements led to the identification of 6-methyl-N-((1R)-1-(4-(trifluoromethoxy)phenyl)propyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (38a), a molecule with the desired balance of preclinical properties. Further characterization by cocrystal structure analysis of 38a bound to PDE2A uncovered a unique binding mode and provided insights into its observed potency and PDE selectivity. Compound 38a significantly elevated 3',5'-cyclic guanosine monophosphate (cGMP) levels in mouse brain following oral administration, thus validating this compound as a useful pharmacological tool and an attractive lead for future optimization.