A High-throughput Response to the SARS-CoV-2 Pandemic.

Four years after the beginning of the COVID-19 pandemic, it is important to reflect on the events that have occurred during that time and the knowledge that has been gained. The response to the pandemic was rapid and highly resourced; it was also built upon a foundation of decades of federally funded basic and applied research. Laboratories in government, pharmaceutical, academic, and non-profit institutions all played roles in advancing pre-2020 discoveries to produce clinical treatments. This perspective provides a summary of how the development of high-throughput screening methods in a biosafety level 3 (BSL-3) environment at Southern Research Institute (SR) contributed to pandemic response efforts. The challenges encountered are described, including those of a technical nature as well as those of working under the pressures of an unpredictable virus and pandemic.

High-Throughput cell-based immunofluorescence assays against influenza.

A rapid drug discovery response to influenza outbreaks with the potential to reach pandemic status could help minimize the virus's impact by reducing the time to identify anti-influenza drugs. Although several anti-influenza strategies have been considered in the search for new drugs, only a few therapeutic agents are approved for clinical use. The cytopathic effect induced by the influenza virus in Madin Darby canine kidney (MDCK) cells has been widely used for high-throughput anti-influenza drug screening, but the fact that the MDCK cells are not human cells constitutes a disadvantage when searching for new therapeutic agents for human use. We have developed a highly sensitive cell-based imaging assay for the identification of inhibitors of influenza A and B virus that is high-throughput compatible using the A549 human cell line. The assay has also been optimized for the assessment of the neutralizing effect of anti-influenza antibodies in the absence of trypsin, which allows testing of purified antibodies and serum samples. This assay platform can be applied to full high-throughput screening campaigns or later stages requiring quantitative potency determinations for structure-activity relationships.

De novo sequencing and construction of a unique antibody for the recognition of alternative conformations of cytochrome c in cells.

Cytochrome c (cyt c) can undergo reversible conformational changes under biologically relevant conditions. Revealing these alternative cyt c conformers at the cell and tissue level is challenging. A monoclonal antibody (mAb) identifying a key conformational change in cyt c was previously reported, but the hybridoma was rendered nonviable. To resurrect the mAb in a recombinant form, the amino-acid sequences of the heavy and light chains were determined by peptide mapping-mass spectrometry-bioinformatic analysis and used to construct plasmids encoding the full-length chains. The recombinant mAb (R1D3) was shown to perform similarly to the original mAb in antigen-binding assays. The mAb bound to a variety of oxidatively modified cyt c species (e.g., nitrated at Tyr74 or oxidized at Met80), which lose the sixth heme ligation (Fe-Met80); it did not bind to several cyt c phospho- and acetyl-mimetics. Peptide competition assays together with molecular dynamic studies support that R1D3 binds a neoepitope within the loop 40-57. R1D3 was employed to identify alternative conformations of cyt c in cells under oxidant- or senescence-induced challenge as confirmed by immunocytochemistry and immunoaffinity studies. Alternative conformers translocated to the nuclei without causing apoptosis, an observation that was further confirmed after pinocytic loading of oxidatively modified cyt c to B16-F1 cells. Thus, alternative cyt c conformers, known to gain peroxidatic function, may represent redox messengers at the cell nuclei. The availability and properties of R1D3 open avenues of interrogation regarding the presence and biological functions of alternative conformations of cyt c in mammalian cells and tissues.

Discovery of a potent M5 antagonist with improved clearance profile. Part 2: Pyrrolidine amide-based antagonists.

This Letter describes our ongoing effort to improve the clearance of selective M5 antagonists. Herein, we report the replacement of the previously disclosed piperidine amide (4, disclosed in Part 1) with a pyrrolidine amide core. Several compounds within this series provided good potency, subtype selectivity, and low to moderate clearance profiles. Interestingly, the left-hand side SAR for this series diverged from our earlier efforts.

Discovery of a potent M5 antagonist with improved clearance profile. Part 1: Piperidine amide-based antagonists.

The lack of potent and selective tool compounds with pharmaceutically favorable properties limits the in vivo understanding of muscarinic acetylcholine receptor subtype 5 (M5) biology. Previously, we presented a highly potent and selective M5 antagonist VU6019650 with a suboptimal clearance profile as our second-generation tool compound. Herein, we disclose our ongoing efforts to generate next-generation M5 antagonists with improved clearance profiles. A mix and match approach between VU6019650 (lead) and VU0500325 (HTS hit) generated a piperidine amide-based novel M5 antagonist series. Several analogs within this series, including 29f, provided good on-target potency with improved clearance profiles, though room for improvement remains.

A Novel Approach To Identify Inhibitors of Iron Acquisition Systems of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen that has been declared by the World Health Organization as a "priority 1 critical pathogen" needing immediate new strategies for chemotherapy. During infection, P. aeruginosa uses redundant mechanisms to acquire ferric, heme (Hm), or ferrous iron from the host to survive and colonize. Significant efforts have been undertaken to develop siderophore blockers to inhibit ferric iron acquisition by P. aeruginosa, but there is a lack of inhibitors that can block Hm or ferrous iron acquisition by P. aeruginosa. We developed and employed a targeted high-throughput screen (HTS) and identified a molecule(s) that can specifically inhibit the Hm and ferrous iron acquisition systems of P. aeruginosa. Our targeted approach relies on screening a small-molecule library against P. aeruginosa under three growth conditions, where the only variable was the iron source (ferric, Hm, or ferrous iron). Each condition served as a counterscreen for the other, and we identified molecules that inhibit the growth of P. aeruginosa in the presence of only Hm or ferrous iron. Our data indicate that econazole, bithionate, and raloxifene inhibit the growth of P. aeruginosa in the presence of Hm and that oxyquinoline inhibits the growth of P. aeruginosa in the presence of ferrous iron. These iron-specific inhibitors do not interfere with the activity of meropenem, a commercial antipseudomonal, and can also increase meropenem activity. In conclusion, we present a proof of concept of a successful targeted conditional screening method by which we can identify specific iron acquisition inhibitors. This approach is highly adaptable and can easily be extended to any other pathogen. IMPORTANCE Since acquiring iron is paramount to P. aeruginosa's survival and colonization in the human host, developing novel strategies to block the access of P. aeruginosa to host iron will allow us to starve it of an essential nutrient. P. aeruginosa uses siderophore, heme, or ferrous iron uptake systems to acquire iron in the human host. We have developed a novel approach through which we can directly identify molecules that can prevent P. aeruginosa from utilizing heme or ferrous iron. This approach overcomes the need for the in silico design of molecules and identifies structurally diverse biologically active inhibitor molecules. This screening approach is adaptable and can be extended to any pathogen. Since Gram-negative pathogens share many similarities in iron acquisition at both the mechanistic and molecular levels, our screening approach presents a significant opportunity to develop novel broad-spectrum iron acquisition inhibitors of Gram-negative pathogens.

Large-Scale Identification of Multiple Classes of Host Defense Peptide-Inducing Compounds for Antimicrobial Therapy.

The rapid emergence of antibiotic resistance demands new antimicrobial strategies that are less likely to develop resistance. Augmenting the synthesis of endogenous host defense peptides (HDPs) has been proven to be an effective host-directed therapeutic approach. This study aimed to identify small-molecule compounds with a strong ability to induce endogenous HDP synthesis for further development as novel antimicrobial agents. By employing a stable HDP promoter-driven luciferase reporter cell line known as HTC/AvBD9-luc, we performed high-throughput screening of 5002 natural and synthetic compounds and identified 110 hits with a minimum Z-score of 2.0. Although they were structurally and functionally diverse, half of these hits were inhibitors of class I histone deacetylases, the phosphoinositide 3-kinase pathway, ion channels, and dopamine and serotonin receptors. Further validations revealed mocetinostat, a benzamide histone deacetylase inhibitor, to be highly potent in enhancing the expression of multiple HDP genes in chicken macrophage cell lines and jejunal explants. Importantly, mocetinostat was more efficient than entinostat and tucidinostat, two structural analogs, in promoting HDP gene expression and the antibacterial activity of chicken macrophages. Taken together, mocetinostat, with its ability to enhance HDP synthesis and the antibacterial activity of host cells, could be potentially developed as a novel antimicrobial for disease control and prevention.

A BRET Ca2+ sensor enables high-throughput screening in the presence of background fluorescence.

The intrinsic fluorescence of samples confounds the use of fluorescence-based sensors. This is of particular concern in high-throughput screening (HTS) applications using large chemical libraries containing intrinsically fluorescent compounds. To overcome this problem, we developed a bioluminescence resonance energy transfer (BRET) Ca2+ sensor, CalfluxCTN. We demonstrated that it reliably reported changes in intracellular Ca2+ concentrations evoked by an agonist and an antagonist of the human muscarinic acetylcholine receptor M1 (hM1R) even in the presence of the fluorescent compound fluorescein, which interfered with a standard fluorescent HTS sensor (Fluo-8). In an HTS using a chemical library containing fluorescent compounds, CalfluxCTN accurately identified agonists and antagonists that were missed or miscategorized using Fluo-8. Moreover, we showed that a luciferase substrate that becomes activated only when inside cells generated long-lasting BRET signals in HTS, enabling results to be reliably compared among replicate samples for hours. Thus, the use of a self-luminescent sensor instead of a fluorescent sensor could facilitate the complete screening of chemical libraries in a high-throughput context and enable analysis of autofluorescent samples in many different applications.

Development of VU6019650: A Potent, Highly Selective, and Systemically Active Orthosteric Antagonist of the M5 Muscarinic Acetylcholine Receptor for the Treatment of Opioid Use Disorder.

The muscarinic acetylcholine receptor (mAChR) subtype 5 (M5) represents a novel potential target for the treatment of multiple addictive disorders, including opioid use disorder. Through chemical optimization of several functional high-throughput screening hits, VU6019650 (27b) was identified as a novel M5 orthosteric antagonist with high potency (human M5 IC50 = 36 nM), M5 subtype selectivity (>100-fold selectivity against human M1-4) and favorable physicochemical properties for systemic dosing in preclinical addiction models. In acute brain slice electrophysiology studies, 27b blocked the nonselective muscarinic agonist oxotremorine-M-induced increases in neuronal firing rates of midbrain dopamine neurons in the ventral tegmental area, a part of the mesolimbic dopaminergic reward circuitry. Moreover, 27b also inhibited oxycodone self-administration in male Sprague-Dawley rats within a dose range that did not impair general motor output.

Structure based virtual screening identifies small molecule effectors for the sialoglycan binding protein Hsa.

Infective endocarditis (IE) is a cardiovascular disease often caused by bacteria of the viridans group of streptococci, which includes Streptococcus gordonii and Streptococcus sanguinis. Previous research has found that serine-rich repeat (SRR) proteins on the S. gordonii bacterial surface play a critical role in pathogenesis by facilitating bacterial attachment to sialylated glycans displayed on human platelets. Despite their important role in disease progression, there are currently no anti-adhesive drugs available on the market. Here, we performed structure-based virtual screening using an ensemble docking approach followed by consensus scoring to identify novel small molecule effectors against the sialoglycan binding domain of the SRR adhesin protein Hsa from the S. gordonii strain DL1. The screening successfully predicted nine compounds which were able to displace the native ligand (sialyl-T antigen) in an in vitro assay and bind competitively to Hsa. Furthermore, hierarchical clustering based on the MACCS fingerprints showed that eight of these small molecules do not share a common scaffold with the native ligand. This study indicates that SRR family of adhesin proteins can be inhibited by diverse small molecules and thus prevent the interaction of the protein with the sialoglycans. This opens new avenues for discovering potential drugs against IE.

Symmetrically substituted dichlorophenes inhibit N-acyl-phosphatidylethanolamine phospholipase D.

N-Acyl-phosphatidylethanolamine phospholipase D (NAPE-PLD) (EC 3.1.4.4) catalyzes the final step in the biosynthesis of N-acyl-ethanolamides. Reduced NAPE-PLD expression and activity may contribute to obesity and inflammation, but a lack of effective NAPE-PLD inhibitors has been a major obstacle to elucidating the role of NAPE-PLD and N-acyl-ethanolamide biosynthesis in these processes. The endogenous bile acid lithocholic acid (LCA) inhibits NAPE-PLD activity (with an IC50 of 68 µm), but LCA is also a highly potent ligand for TGR5 (EC50 0.52 µm). Recently, the first selective small-molecule inhibitor of NAPE-PLD, ARN19874, has been reported (having an IC50 of 34 µm). To identify more potent inhibitors of NAPE-PLD, here we used a quenched fluorescent NAPE analog, PED-A1, as a substrate for recombinant mouse Nape-pld to screen a panel of bile acids and a library of experimental compounds (the Spectrum Collection). Muricholic acids and several other bile acids inhibited Nape-pld with potency similar to that of LCA. We identified 14 potent Nape-pld inhibitors in the Spectrum Collection, with the two most potent (IC50 = ∼2 µm) being symmetrically substituted dichlorophenes, i.e. hexachlorophene and bithionol. Structure-activity relationship assays using additional substituted dichlorophenes identified key moieties needed for Nape-pld inhibition. Both hexachlorophene and bithionol exhibited significant selectivity for Nape-pld compared with nontarget lipase activities such as Streptomyces chromofuscus PLD or serum lipase. Both also effectively inhibited NAPE-PLD activity in cultured HEK293 cells. We conclude that symmetrically substituted dichlorophenes potently inhibit NAPE-PLD in cultured cells and have significant selectivity for NAPE-PLD versus other tissue-associated lipases.

A Robust and Scalable High-Throughput Compatible Assay for Screening Amyloid-ß-Binding Compounds.

A robust fluorescent readout assay using topologically-sensitive dyes improves the screening of novel amyloid-binding molecules. One of the key components that make this assay more realistic is the use of endogenous amyloid obtained from 5XFAD mouse brains. The assay conditions were optimized for high throughput screening operation with Z-prime values >0.6. Using a combination of library of 3,500 compounds including known drugs, natural-derived molecules and random organic molecules, 8 unique molecules were identified as potential amyloid-binding agents.

Comparative analysis of myometrial and vascular smooth muscle cells to determine optimal cells for use in drug discovery.

Novel therapeutic regulators of uterine contractility are needed to manage preterm labor, induce labor and control postpartum hemorrhage. Therefore, we previously developed a high-throughput assay for large-scale screening of small molecular compounds to regulate calcium-mobilization in primary mouse uterine myometrial cells. The goal of this study was to select the optimal myometrial cells for our high-throughput drug discovery assay, as well as determine the similarity or differences of myometrial cells to vascular smooth muscle cells (VSMCs)-the most common off-target of current myometrial therapeutics. Molecular and pharmacological assays were used to compare myometrial cells from four sources: primary cells isolated from term pregnant human and murine myometrium, immortalized pregnant human myometrial (PHM-1) cells and immortalized non-pregnant human myometrial (hTERT-HM) cells. In addition, myometrial cells were compared to vascular SMCs. We found that the transcriptome profiles of hTERT-HM and PHM1 cells were most similar (r = 0.93 and 0.90, respectively) to human primary myometrial cells. Comparative transcriptome profiling of primary human myometrial transcriptome and VSMCs revealed 498 upregulated (p ≤ 0.01, log2FC≥1) genes, of which 142 can serve as uterine-selective druggable targets. In the high-throughput Ca2+-assay, PHM1 cells had the most similar response to primary human myometrial cells in OT-induced Ca2+-release (Emax = 195% and 143%, EC50 = 30 nM and 120 nM, respectively), while all sources of myometrial cells showed excellent and similar robustness and reproducibility (Z' = 0.52 to 0.77). After testing a panel of 61 compounds, we found that the stimulatory and inhibitory responses of hTERT-HM cells were highly-correlated (r = 0.94 and 0.95, respectively) to human primary cells. Moreover, ten compounds were identified that displayed uterine-selectivity (≥5-fold Emax or EC50 compared to VSMCs). Collectively, this study found that hTERT-HM cells exhibited the most similarity to primary human myometrial cells and, therefore, is an optimal substitute for large-scale screening to identify novel therapeutic regulators of myometrial contractility. Moreover, VSMCs can serve as an important counter-screening tool to assess uterine-selectivity of targets and drugs given the similarity observed in the transcriptome and response to compounds.

Challenges in the Discovery and Optimization of mGlu2/4 Heterodimer Positive Allosteric Modulators.

BACKGROUND: This article describes the challenges in the discovery and optimization of mGlu2/4 heterodimer Positive Allosteric Modulators (PAMs). METHODS: Initial forays based on VU0155041, a PAM of both the mGlu4 homodimer and the mGlu2/4 heterodimer, led to flat, intractable SAR that precluded advancement. Screening of a collection of 1,152 FDA approved drugs led to the discovery that febuxostat, an approved xanthine oxidase inhibitor, was a moderately potent PAM of the mGlu2/4 heterodimer (EC50 = 3.4 µM), but was peripherally restricted (rat Kp = 0.03). Optimization of this hit led to PAMs with improved potency (EC50s <800 nM) and improved CNS penetration (rat Kp >2, an ~100-fold increase). RESULTS: However, these new amide analogs of febuxostat proved to be either GIRK1/2 and GIRK1/4 activators (primary carboxamide congeners) or mGlu2 PAMs (secondary and tertiary amides) and not selective mGlu2/4 heterodimer PAMs. CONCLUSION: These results required the team to develop a new screening cascade paradigm, and exemplified the challenges in developing allosteric ligands for heterodimeric receptors.

Discovery, Characterization, and Effects on Renal Fluid and Electrolyte Excretion of the Kir4.1 Potassium Channel Pore Blocker, VU0134992.

The inward rectifier potassium (Kir) channel Kir4.1 (KCNJ10) carries out important physiologic roles in epithelial cells of the kidney, astrocytes in the central nervous system, and stria vascularis of the inner ear. Loss-of-function mutations in KCNJ10 lead to EAST/SeSAME syndrome, which is characterized by epilepsy, ataxia, renal salt wasting, and sensorineural deafness. Although genetic approaches have been indispensable for establishing the importance of Kir4.1 in the normal function of these tissues, the availability of pharmacological tools for acutely manipulating the activity of Kir4.1 in genetically normal animals has been lacking. We therefore carried out a high-throughput screen of 76,575 compounds from the Vanderbilt Institute of Chemical Biology library for small-molecule modulators of Kir4.1. The most potent inhibitor identified was 2-(2-bromo-4-isopropylphenoxy)-N-(2,2,6,6-tetramethylpiperidin-4-yl)acetamide (VU0134992). In whole-cell patch-clamp electrophysiology experiments, VU0134992 inhibits Kir4.1 with an IC50 value of 0.97 µM and is 9-fold selective for homomeric Kir4.1 over Kir4.1/5.1 concatemeric channels (IC50 = 9 µM) at -120 mV. In thallium (Tl+) flux assays, VU0134992 is greater than 30-fold selective for Kir4.1 over Kir1.1, Kir2.1, and Kir2.2; is weakly active toward Kir2.3, Kir6.2/SUR1, and Kir7.1; and is equally active toward Kir3.1/3.2, Kir3.1/3.4, and Kir4.2. This potency and selectivity profile is superior to Kir4.1 inhibitors amitriptyline, nortriptyline, and fluoxetine. Medicinal chemistry identified components of VU0134992 that are critical for inhibiting Kir4.1. Patch-clamp electrophysiology, molecular modeling, and site-directed mutagenesis identified pore-lining glutamate 158 and isoleucine 159 as critical residues for block of the channel. VU0134992 displayed a large free unbound fraction (fu) in rat plasma (fu = 0.213). Consistent with the known role of Kir4.1 in renal function, oral dosing of VU0134992 led to a dose-dependent diuresis, natriuresis, and kaliuresis in rats. Thus, VU0134992 represents the first in vivo active tool compound for probing the therapeutic potential of Kir4.1 as a novel diuretic target for the treatment of hypertension.

Screening for AMPA receptor auxiliary subunit specific modulators.

AMPA receptors (AMPAR) are ligand gated ion channels critical for synaptic transmission and plasticity. Their dysfunction is implicated in a variety of psychiatric and neurological diseases ranging from major depressive disorder to amyotrophic lateral sclerosis. Attempting to potentiate or depress AMPAR activity is an inherently difficult balancing act between effective treatments and debilitating side effects. A newly explored strategy to target subsets of AMPARs in the central nervous system is to identify compounds that affect specific AMPAR-auxiliary subunit complexes. This exploits diverse spatio-temporal expression patterns of known AMPAR auxiliary subunits, providing means for designing brain region-selective compounds. Here we report a high-throughput screening-based pipeline that can identify compounds that are selective for GluA2-CNIH3 and GluA2-stargazin complexes. These compounds will help us build upon the growing library of AMPAR-auxiliary subunit specific inhibitors, which have thus far all been targeted to TARP γ-8. We used a cell-based assay combined with a voltage-sensitive dye (VSD) to identify changes in glutamate-gated cation flow across the membranes of HEK cells co-expressing GluA2 and an auxiliary subunit. We then used a calcium flux assay to further validate hits picked from the VSD assay. VU0612951 and VU0627849 are candidate compounds from the initial screen that were identified as negative and positive allosteric modulators (NAM and PAM), respectively. They both have lower IC50/EC50s on complexes containing stargazin and CNIH3 than GSG1L or the AMPAR alone. We have also identified a candidate compound, VU0539491, that has NAM activity in GluA2(R)-CNIH3 and GluA2(Q) complexes and PAM activity in GluA2(Q)-GSG1L complexes.

Selective Small Molecule Activators of TREK-2 Channels Stimulate Dorsal Root Ganglion c-Fiber Nociceptor Two-Pore-Domain Potassium Channel Currents and Limit Calcium Influx.

The two-pore-domain potassium (K2P) channel TREK-2 serves to modulate plasma membrane potential in dorsal root ganglia c-fiber nociceptors, which tunes electrical excitability and nociception. Thus, TREK-2 channels are considered a potential therapeutic target for treating pain; however, there are currently no selective pharmacological tools for TREK-2 channels. Here we report the identification of the first TREK-2 selective activators using a high-throughput fluorescence-based thallium (Tl+) flux screen (HTS). An initial pilot screen with a bioactive lipid library identified 11-deoxy prostaglandin F2α as a potent activator of TREK-2 channels (EC50 ≈ 0.294 µM), which was utilized to optimize the TREK-2 Tl+ flux assay (Z' = 0.752). A HTS was then performed with 76 575 structurally diverse small molecules. Many small molecules that selectively activate TREK-2 were discovered. As these molecules were able to activate single TREK-2 channels in excised membrane patches, they are likely direct TREK-2 activators. Furthermore, TREK-2 activators reduced primary dorsal root ganglion (DRG) c-fiber Ca2+ influx. Interestingly, some of the selective TREK-2 activators such as 11-deoxy prostaglandin F2α were found to inhibit the K2P channel TREK-1. Utilizing chimeric channels containing portions of TREK-1 and TREK-2, the region of the TREK channels that allows for either small molecule activation or inhibition was identified. This region lies within the second pore domain containing extracellular loop and is predicted to play an important role in modulating TREK channel activity. Moreover, the selective TREK-2 activators identified in this HTS provide important tools for assessing human TREK-2 channel function and investigating their therapeutic potential for treating chronic pain.

Delayed treatment with PTBA analogs reduces postinjury renal fibrosis after kidney injury.

No therapies have been shown to accelerate recovery or prevent fibrosis after acute kidney injury (AKI). In part, this is because most therapeutic candidates have to be given at the time of injury and the diagnosis of AKI is usually made too late for drugs to be efficacious. Strategies to enhance post-AKI repair represent an attractive approach to address this. Using a phenotypic screen in zebrafish, we identified 4-(phenylthio)butanoic acid (PTBA), which promotes proliferation of embryonic kidney progenitor cells (EKPCs), and the PTBA methyl ester UPHD25, which also increases postinjury repair in ischemia-reperfusion and aristolochic acid-induced AKI in mice. In these studies, a new panel of PTBA analogs was evaluated. Initial screening was performed in zebrafish EKPC assays followed by survival assays in a gentamicin-induced AKI larvae zebrafish model. Using this approach, we identified UPHD186, which in contrast to UPHD25, accelerates recovery and reduces fibrosis when administered several days after ischemia-reperfusion AKI and reduces fibrosis after unilateral ureteric obstruction in mice. UPHD25 and 186 are efficiently metabolized to the active analog PTBA in liver and kidney microsome assays, indicating both compounds may act as PTBA prodrugs in vivo. UPHD186 persists longer in the circulation than UPHD25, suggesting that sustained levels of UPHD186 may increase efficacy by acting as a reservoir for renal metabolism to PTBA. These findings validate use of zebrafish EKPC and AKI assays as a drug discovery strategy for molecules that reduce fibrosis in multiple AKI models and can be administered days after initiation of injury.

Acyl dihydropyrazolo[1,5-a]pyrimidinones as metabotropic glutamate receptor 5 positive allosteric modulators.

We report the optimization of a series of metabotropic glutamate receptor 5 (mGlu5) positive allosteric modulators (PAMs) from an acyl dihydropyrazolo[1,5-a]pyrimidinone class. Investigation of exocyclic amide transpositions with this unique 5,6-bicyclic core were conducted in attempt to modulate physicochemical properties and identify a suitable backup candidate with a reduced half-life. A potent and selective PAM, 1-(2-(phenoxymethyl)-6,7-dihydropyrazolo[1,5-a]pyrimidin-4(5H)-yl)ethanone (9a, VU0462807), was identified with superior solubility and efficacy in the acute amphetamine-induced hyperlocomotion (AHL) rat model with a minimum effective dose of 3mg/kg. Attempts to mitigate oxidative metabolism of the western phenoxy of 9a through extensive modification and profiling are described.

Discovery of VU0409551/JNJ-46778212: An mGlu5 Positive Allosteric Modulator Clinical Candidate Targeting Schizophrenia.

Herein, we report the structure-activity relationship of a novel series of (2(phenoxymethyl)-6,7-dihydrooxazolo[5,4-c]pyridine-5(4H)-yl(aryl)methanones as potent, selective, and orally bioavailable metabotropic glutamate receptor subtype 5 (mGlu5) positive allosteric modulators (PAMs). On the basis of its robust in vitro potency and in vivo efficacy in multiple preclinical models of multiple domains of schizophrenia, coupled with a good DMPK profile and an acceptable therapeutic window, 17a (VU0409551/JNJ-46778212) was selected as a candidate for further development.