Metabolic Profiling of S-praziquantel: Structure Elucidation Using the Crystalline Sponge Method in Combination with Mass Spectrometry and Nuclear Magnetic Resonance.

Praziquantel (PZQ) is the drug of choice for treatment of the neglected tropical disease schistosomiasis. Although the drug has been extensively used over several decades and its metabolism well studied (several oxidative metabolites are known from literature), the knowledge of the complete structure of some of its metabolites remains elusive. Conventional techniques, such as nuclear magnetic resonance or liquid chromatography mass spectrometry were used in the past to investigate phase I and phase II metabolites of PZQ. These techniques are either limited to provide the complete molecular structure (liquid chromatography mass spectrometry) or require large amount of sample material (NMR), which are not always available when in vitro systems are used for investigation of the metabolites. In this study, we describe new structures of S-PZQ metabolites generated in vitro from human liver microsomes using the crystalline sponge method. After chromatographic separation and purification of the oxidative metabolites, ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry analysis was conducted to narrow down the position of oxidation to a certain part of the molecule. To determine the exact position of hydroxylation, singe-crystal X-ray diffraction analysis of the crystalline sponges and absorbed analyte was used to identify the structure of S-PZQ and its metabolites. The crystalline sponge method allowed for complete structure elucidation of the known metabolites S-trans-4'-hydroxy-PZQ (M1), S-cis-4'-hydroxy-PZQ (M2) and S-/R-11b-hydroxy-PZQ (M6) as well as the unknown metabolites S-9-hydroxy-PZQ (M3) and S-7-hydroxy-S-PZQ (M4). For comparison of structural elucidation techniques, one metabolite (M3) was additionally analyzed using NMR. SIGNIFICANCE STATEMENT: The information content of the metabolic pathway of praziquantel is still limited. The crystalline sponge method allowed the complete structural elucidation of three known and two unknown metabolites of S-praziquantel, using only trace amounts of analyte material, as demonstrated in this study.

Translation of liver stage activity of M5717, a Plasmodium elongation factor 2 inhibitor: from bench to bedside.

BACKGROUND: Targeting the asymptomatic liver stage of Plasmodium infection through chemoprevention could become a key intervention to reduce malaria-associated incidence and mortality. METHODS: M5717, a Plasmodium elongation factor 2 inhibitor, was assessed in vitro and in vivo with readily accessible Plasmodium berghei parasites. In an animal refinement, reduction, replacement approach, the in vitro IC99 value was used to feed a Population Pharmacokinetics modelling and simulation approach to determine meaningful effective doses for a subsequent Plasmodium sporozoite-induced volunteer infection study. RESULTS: Doses of 100 and 200 mg would provide exposures exceeding IC99 in 96 and 100% of the simulated population, respectively. CONCLUSIONS: This approach has the potential to accelerate the search for new anti-malarials, to reduce the number of healthy volunteers needed in a clinical study and decrease and refine the animal use in the preclinical phase.

Deconvolution of Cytochrome P450 Induction Mechanisms in HepaRG Nuclear Hormone Receptor Knockout Cells.

Pregnane X receptor (PXR), constitutive androstane receptor (CAR), and PXR/CAR knockout (KO) HepaRG cells, as well as a PXR reporter gene assay, were used to investigate the mechanism of CYP3A4 and CYP2B6 induction by prototypical substrates and a group of compounds from the Merck KGaA oncology drug discovery pipeline. The basal and inducible gene expression of CYP3A4 and CYP2B6 of nuclear hormone receptor (NHR) KO HepaRG relative to control HepaRG was characterized. The basal expression of CYP3A4 was markedly higher in the PXR (10-fold) and CAR (11-fold) KO cell lines compared with control HepaRG, whereas inducibility was substantially lower. Inversely, basal expression of CYP3A4 in PXR/CAR double KO (dKO) was low (10-fold reduction). Basal CYP2B6 expression was high in PXR KO (9-fold) cells which showed low inducibility, whereas the basal expression remained unchanged in CAR and dKO cell lines compared with control cells. Most of the test compounds induced CYP3A4 and CYP2B6 via PXR and, to a lesser extent, via CAR. Furthermore, other non-NHR-driven induction mechanisms were implicated, either alone or in addition to NHRs. Notably, 5 of the 16 compounds (31%) that were PXR inducers in HepaRG did not activate PXR in the reporter gene assay, illustrating the limitations of this system. This study indicates that HepaRG is a highly sensitive system fit for early screening of cytochrome P450 (P450) induction in drug discovery. Furthermore, it shows the applicability of HepaRG NHR KO cells as tools to deconvolute mechanisms of P450 induction using novel compounds representative for oncology drug discovery. SIGNIFICANCE STATEMENT: This work describes the identification of induction mechanisms of CYP3A4 and CYP2B6 for an assembly of oncology drug candidates using HepaRG nuclear hormone receptor knockout and displays its advantages compared to a pregnane X receptor reporter gene assay. With this study, risk assessment of drug candidates in early drug development can be improved.

Preclinical Antimalarial Combination Study of M5717, a Plasmodium falciparum Elongation Factor 2 Inhibitor, and Pyronaridine, a Hemozoin Formation Inhibitor.

Antimalarial drug resistance in the Plasmodium falciparum parasite poses a constant challenge for drug development. To mitigate this risk, new antimalarial medicines should be developed as fixed-dose combinations. Assessing the pharmacodynamic interactions of potential antimalarial drug combination partners during early phases of development is essential in developing the targeted parasitological and clinical profile of the final drug product. Here, we have studied the combination of M5717, a P. falciparum translation elongation factor 2 inhibitor, and pyronaridine, an inhibitor of hemozoin formation. Our test cascade consisted of in vitro isobolograms as well as in vivo studies in the P. falciparum severe combined immunodeficient (SCID) mouse model. We also analyzed pharmacokinetic and pharmacodynamic parameters, including genomic sequencing of recrudescent parasites. We observed no pharmacokinetic interactions with the combination of M5717 and pyronaridine. M5717 did not negatively impact the rate of kill of the faster-acting pyronaridine, and the latter was able to suppress the selection of M5717-resistant mutants, as well as significantly delay the recrudescence of parasites both with suboptimal and optimal dosing regimens.

Crystalline Sponges as a Sensitive and Fast Method for Metabolite Identification: Application to Gemfibrozil and its Phase I and II Metabolites.

Understanding the metabolism of new drug candidates is important during drug discovery and development, as circulating metabolites may contribute to efficacy or cause safety issues. In the early phase of drug discovery, human in vitro systems are used to investigate human relevant metabolism. Though conventional techniques are limited in their ability to provide complete molecular structures of metabolites (liquid chromatography mass spectrometry) or require a larger amount of material not available from in vitro incubation (nuclear magnetic resonance), we here report for the first time the use of the crystalline sponge method to identify phase I and phase II metabolites generated from in vitro liver microsomes or S9 fractions. Gemfibrozil was used as a test compound. Metabolites generated from incubation with microsomes or S9 fractions, were fractionated using online fraction collection. After chromatographic purification and fractionation of the generated metabolites, single crystal X-ray diffraction of crystalline sponges was used to identify the structure of gemfibrozil metabolites. This technique allowed for complete structure elucidation of 5'-CH2OH gemfibrozil (M1), 4'-OH gemfibrozil (M2), 5'-COOH gemfibrozil (M3), and the acyl glucuronide of gemfibrozil, 1-O-ß-glucuronide (M4), the first acyl glucuronide available in the Cambridge Crystallographic Data Centre. Our study shows that when optimal soaking is possible, crystalline sponges technology is a sensitive (nanogram amount) and fast (few days) method that can be applied early in drug discovery to identify the structure of pure metabolites from in vitro incubations. SIGNIFICANCE STATEMENT: Complete structure elucidation of human metabolites plays a critical role in early drug discovery. Low amounts of material (nanogram) are only available at this stage and insufficient for nuclear magnetic resonance analysis. The crystalline sponge method has the potential to close this gap, as demonstrated in this study.

Prediction of volume of distribution in humans: analysis of eight methods and their application in drug discovery.

The performance of eight different methods to predict human volume of distribution (VDss) using a large data set (N > 100) was evaluated.The accuracy was assessed by the end points % within two-fold and absolute average fold error (AAFE). The ability to rank order was accessed by the σ and bias was examined using average fold error. Significance of observed differences was established using statistical permutation testing.The Rodgers-Lukova equation, a tissue composition model, for acids and single species scaling based on rat for other ion classes showed the best results in absence of non-rodent data.The semimechanistic Øie-Tozer model based on all thee preclinical species showed the best performance overall (81% within two-fold, AAFE 1.55, σ 0.62). This was not statistically significantly better at the 95% confidence level than the same model based on two preclinical species or single species scaling from monkey. Thus, the use of primates appears difficult to justify when the sole goal is to extrapolate human volume of distribution.

Improved Prediction of in Vivo Supersaturation and Precipitation of Poorly Soluble Weakly Basic Drugs Using a Biorelevant Bicarbonate Buffer in a Gastrointestinal Transfer Model.

The characterization of intestinal dissolution of poorly soluble drugs represents a key task during the development of both new drug candidates and drug products. The bicarbonate buffer is considered as the most biorelevant buffer for simulating intestinal conditions. However, because of its complex nature, being the volatility of CO2, it has only been rarely used in the past. The aim of this study was to investigate the effect of a biorelevant bicarbonate buffer on intestinal supersaturation and precipitation of poorly soluble drugs using a gastrointestinal (GI) transfer model. Therefore, the results of ketoconazole, pazopanib, and lapatinib transfer model experiments using FaSSIFbicarbonate were compared with the results obtained using standard FaSSIFphosphate. Additionally, the effect of hydroxypropyl methylcellulose acetate succinate (HPMCAS) as a precipitation inhibitor was investigated in both buffer systems and compared to rat pharmacokinetic (PK) studies with and without coadministration of HPMCAS as a precipitation inhibitor. While HPMCAS was found to be an effective precipitation inhibitor for all drugs in FaSSIFphosphate, the effect in FaSSIFbicarbonate was much less pronounced. The PK studies revealed that HPMCAS did not increase the exposure of any of the model compounds significantly, indicating that the transfer model employing bicarbonate-buffered FaSSIF has a better predictive power compared to the model using phosphate-buffered FaSSIF. Hence, the application of a bicarbonate buffer in a transfer model set-up represents a promising approach to increase the predictive power of this in vitrotool and to contribute to the development of drug substances and drug products in a more biorelevant way.

Parkinson's disease-like burst firing activity in subthalamic nucleus induced by AAV-α-synuclein is normalized by LRRK2 modulation.

Parkinson's disease (PD) affects motor function through degenerative processes and synaptic transmission impairments in the basal ganglia. None of the treatments available delays or stops the progression of the disease. While α-synuclein pathological accumulation represents a hallmark of the disease in its idiopathic form, leucine rich repeat kinase 2 (LRRK2) is genetically associated with familial and sporadic forms of PD. The genetic information suggests that LRRK2 kinase activity plays a role in the pathogenesis of the disease. To support a potential link between LRRK2 and α-synuclein in the pathophysiological mechanisms underlying PD, the effect of LRRK2 ablation or LRRK2 kinase pharmacological inhibition were studied in rats with adeno-associated virus-induced (AAV) α-synuclein overexpression in the nigrostriatal pathway. We first report that viral overexpression of α-synuclein induced increased burst firing in subthalamic neurons. Aberrant firing pattern of subthalamic neurons has also been reported in PD patients and neurotoxin-based animal models, and is hypothesized to play a key role in the appearance of motor dysfunction. We further report that genetic LRRK2 ablation, as well as pharmacological inhibition of LRRK2 kinase activity with PFE-360, reversed the aberrant firing pattern of subthalamic neurons induced by AAV-α-synuclein overexpression. This effect of LRRK2 modulation was not associated with any neuroprotective effect or motor improvement. Nonetheless, our findings may indicate a potential therapeutic benefit of LRRK2 kinase inhibition by normalizing the aberrant neuronal activity of subthalamic neurons induced by AAV-α-synuclein, a neurophysiological trait recapitulating observations in PD.

A small molecule activator of Nav 1.1 channels increases fast-spiking interneuron excitability and GABAergic transmission in vitro and has anti-convulsive effects in vivo.

Nav 1.1 (SCN1A) channels primarily located in gamma-aminobutyric acid (GABA)ergic fast-spiking interneurons are pivotal for action potential generation and propagation in these neurons. Inappropriate function of fast-spiking interneurons, leading to disinhibition of pyramidal cells and network desynchronization, correlates with decreased cognitive capability. Further, reduced functionality of Nav 1.1 channels is linked to various diseases in the central nervous system. There is, at present, however no subtype selective pharmacological activators of Nav 1.1 channels available for studying pharmacological modulation of interneuron function. In the current study, we identified a small molecule Nav 1.1 activator, 3-amino-5-(4-methoxyphenyl)thiophene-2-carboxamide, named AA43279, and provided an in vitro to in vivo characterization of the compound. In HEK-293 cells expressing human Nav 1.1 channels, AA43279 increased the Nav 1.1-mediated current in a concentration-dependent manner mainly by impairing the fast inactivation kinetics of the channels. In rat hippocampal brain slices, AA43279 increased the firing activity of parvalbumin-expressing, fast-spiking GABAergic interneurons and increased the spontaneous inhibitory post-synaptic currents (sIPSCs) recorded from pyramidal neurons. When tested in vivo, AA43279 had anti-convulsive properties in the maximal electroshock seizure threshold test. AA43279 was tested for off-target effects on 72 different proteins, including Nav 1.2, Nav 1.4, Nav 1.5, Nav 1.6 and Nav 1.7 and exhibited reasonable selectivity. Taken together, AA43279 might constitute a valuable tool compound for revealing biological functions of Nav 1.1 channels.

The design and SAR of a novel series of 2-aminopyridine based LRRK2 inhibitors.

Leucine-rich repeat kinase 2 (LRRK2) has attracted considerable interest as a therapeutic target for the treatment of Parkinson's disease. Compounds derived from a 2-aminopyridine screening hit were optimised using a LRRK2 homology model based on mixed lineage kinase 1 (MLK1), such that a 2-aminopyridine-based lead molecule 45, with in vivo activity, was identified.

An invertebrate model for CNS drug discovery: Transcriptomic and functional analysis of a mammalian P-glycoprotein ortholog.

BACKGROUND: ABC efflux transporters at the blood brain barrier (BBB), namely the P-glycoprotein (P-gp), restrain the development of central nervous system (CNS) drugs. Consequently, early screening of CNS drug candidates is pivotal to identify those affected by efflux activity. Therefore, simple, high-throughput and predictive screening models are required. The grasshopper (locust) has been developed as an invertebrate in situ model for BBB permeability assessment, as it has shown similarities to vertebrate models. METHODS: Transcriptome profiling of ABC efflux transporters in the locust brain was performed. Subsequently, identified transcripts were matched with their counterparts in human, rat, mouse and Drosophila melanogaster, based on amino acid sequence similarity, and phylogenetic trees were constructed to reveal the most likely evolutionary history of the proteins. Further, functional characterization of a P-gp ortholog was achieved through transport studies, using a selective P-gp substrate and locust brain in situ, followed by kinetic analyses. RESULTS: A protein with high sequence similarity to the ABCB1 gene of vertebrates was found in the locust brain, which encodes P-gp in human and is considered the most vital efflux pump. Functionally, this model showed transport kinetic behaviors comparable to those obtained from in vitro models. Particularly, substrate affinity of the putative P-gp was observed as in P-gp expressing cells lines, used for predicting drug penetration across biological barriers. CONCLUSION: Findings suggest a conserved mechanism of brain efflux activity between insects and vertebrates, confirming that this model holds promise for inexpensive and high-throughput screening relative to in vivo models, for CNS drug discovery.

Linking HSP90 target occupancy to HSP70 induction and efficacy in mouse brain.

HSP90 (Heat shock protein 90) is a molecular chaperone protein ubiquitously expressed throughout all tissues in the body. HSP90 has been proposed as a target to increase turnover of pathological proteins leading to neurodegeneration in Huntington's disease, Parkinson's disease and Alzheimer's disease. The mechanism of how HSP90 inhibition leads to clearance of misfolded proteins is not fully understood. It may involve direct effects of inhibiting ATPase function, indirect effects by inducing the heat-shock-response resulting in upregulation of other chaperone proteins like HSP70 or a combination of both. In the current work we established a methodology to investigate the relationship between HSP90 target occupancy and HSP70 induction in vivo. We also characterized the acute effect of two different HSP90 inhibitors in the rTg4510 transgenic mouse model of Alzheimer's disease which displays a tau-mediated synaptic dysfunction. We show that reversal of synaptic impairments in this model can be obtained with a compound which has a high HSP70 induction capacity. The current developed assay methodologies may thus be of significant use in the further elucidation of the mechanism involved in the in vivo effect of HSP90 inhibition in models of neurodegeneration. Further on, the ability of HSP90 inhibitors to normalize synaptic dysfunction in an in vivo disease model of Alzheimer's disease could have therapeutic relevance and further strengthens the usefulness of this animal model to establish pharmacodynamic effect of HSP90 inhibition.

Characterization of midazolam metabolism in locusts: the role of a CYP3A4-like enzyme in the formation of 1'-OH and 4-OH midazolam.

1. The metabolism of midazolam was investigated in vivo in locusts in order to evaluate the presence of an enzyme with functionality similar to human CYP3A4/5. 2. Hydroxylated metabolites of midazolam identical to human metabolites were detected in locusts and the apparent affinities (Km values) were in the same range as reported in humans (in locusts: 7-23 and 33-85 µM for the formation of the 1'-OH and 4-OH metabolites, respectively). 3. The formation of hydroxylated metabolites could successfully be inhibited by co-administration of ketoconazole, a known CYP3A4/5 inhibitor. 4. Besides phase I metabolites, a number of conjugated metabolites were detected using high-resolution mass spectrometry. The most abundant metabolites detected were structurally identified by (1)H NMR as two N-glucosides. NMR analysis strongly suggested that the glycosylation occurred at the two nitrogens (either one in each case) of the imidazole ring. 5. Distribution of midazolam and the glucose conjugates were successfully measured using desorption electrospray mass spectrometry imaging revealing time-dependent changes in distribution over time. 6. In conclusion, it appears that an enzyme with functionality similar to human CYP3A4/5 is present in locusts. However, it appears that conjugation with glucose is the main detoxification pathway of midazolam in locusts.

Evaluation of 309 molecules as inducers of CYP3A4, CYP2B6, CYP1A2, OATP1B1, OCT1, MDR1, MRP2, MRP3 and BCRP in cryopreserved human hepatocytes in sandwich culture.

1. Regulation of hepatic metabolism or transport may lead to increase in drug clearance and compromise efficacy or safety. In this study, cryopreserved human hepatocytes were used to assess the effect of 309 compounds on the activity and mRNA expression (using qPCR techniques) of CYP1A2, CYP2B6 and CYP3A4, as well as mRNA expression of six hepatic transport proteins: OATP1B1 (SCLO1B1), OCT1 (SLC22A1), MDR1 (ABCB1), MRP2 (ABCC2), MRP3 (ABCC3) and BCRP (ABCG2). 2. The results showed that 6% of compounds induced CYP1A2 activity (1.5-fold increase); 30% induced CYP2B6 while 23% induced CYP3A4. qPCR data identified 16, 33 or 32% inducers of CYP1A2, CYP2B6 or CYP3A4, respectively. MRP2 was induced by 27 compounds followed by MDR1 (16)>BCRP (9)>OCT1 (8)>OATP1B1 (5)>MRP3 (2). 3. CYP3A4 appeared to be down-regulated (≥2-fold decrease in mRNA expression) by 53 compounds, 10 for CYP2B6, 6 for OCT1, 4 for BCRP, 2 for CYP1A2 and OATP1B1 and 1 for MDR1 and MRP2. 4. Structure-activity relationship analysis showed that CYP2B6 and CYP3A4 inducers are bulky lipophilic molecules with a higher number of heavy atoms and a lower number of hydrogen bond donors. Finally, a strategy for testing CYP inducers in drug discovery is proposed.

Identification of a functional homolog of the mammalian CYP3A4 in locusts.

Insects have been proposed as a new tool in early drug development. It was recently demonstrated that locusts have an efflux transporter localized in the blood-brain barrier (BBB) that is functionally similar to the mammalian P-glycoprotein efflux transporter. Two insect BBB models have been put forward, an ex vivo model and an in vivo model. To use the in vivo model it is necessary to fully characterize the locust as an entire organism with regards to metabolic pathways and excretion rate. In the present study, we have characterized the locust metabolism of terfenadine, a compound that in humans is specific to the cytochrome P450 enzyme 3A4. Using high-resolution mass spectrometry coupled to ultra-high-performance liquid chromatography, we have detected metabolites identical to human metabolites of terfenadine. The formation of human metabolites in locusts was inhibited by ketoconazole, a mammalian CYP3A4 inhibitor, suggesting that the enzyme responsible for the human metabolite formation in locusts is functionally similar to human CYP3A4. Besides the human metabolites of terfenadine, additional metabolites were formed in locusts. These were tentatively identified as phosphate and glucose conjugates. In conclusion, not only may locusts be a model useful for determining BBB permeation, but possibly insects could be used in metabolism investigation. However, extensive characterization of the insect model is necessary to determine its applicability.

The grasshopper: a novel model for assessing vertebrate brain uptake.

The aim of the present study was to develop a blood-brain barrier (BBB) permeability model that is applicable in the drug discovery phase. The BBB ensures proper neural function, but it restricts many drugs from entering the brain, and this complicates the development of new drugs against central nervous system diseases. Many in vitro models have been developed to predict BBB permeability, but the permeability characteristics of the human BBB are notoriously complex and hard to predict. Consequently, one single suitable BBB permeability screening model, which is generally applicable in the early drug discovery phase, does not yet exist. A new refined ex vivo insect-based BBB screening model that uses an intact, viable whole brain under controlled in vitro-like exposure conditions is presented. This model uses intact brains from desert locusts, which are placed in a well containing the compound solubilized in an insect buffer. After a limited time, the brain is removed and the compound concentration in the brain is measured by conventional liquid chromatography-mass spectrometry. The data presented here include 25 known drugs, and the data show that the ex vivo insect model can be used to measure the brain uptake over the hemolymph-brain barrier of drugs and that the brain uptake shows linear correlation with in situ perfusion data obtained in vertebrates. Moreover, this study shows that the insect ex vivo model is able to identify P-glycoprotein (Pgp) substrates, and the model allows differentiation between low-permeability compounds and compounds that are Pgp substrates.

Screening novel CNS drug candidates for P-glycoprotein interactions using the cell line iP-gp: In vitro efflux ratios from iP-gp and MDCK-MDR1 monolayers compared to brain distribution data from mice.

Drug efflux by P-glycoprotein (P-gp, ABCB1) is considered as a major obstacle for brain drug delivery for small molecules. P-gp-expressing cell monolayers are used for screening of new drug candidates during early states of drug development. It is, however, uncertain how well the in vitro studies can predict the in vivo P-gp mediated efflux at the blood-brain barrier (BBB). We previously developed a novel cell line of porcine origin, the iP-gp cell line, with high transepithelial resistance and functional expression of human P-gp. The aim of the present study was to evaluate the applicability of the cell line for screening of P-gp interactions of novel drug candidates. For this purpose, bidirectional fluxes of 14 drug candidates were measured in iP-gp cells and in MDCK-MDR1 cells, and compared with pharmacokinetic data obtained in male C57BL/6 mice. The iP-gp cells formed extremely tight monolayers (>15 000 Ω∙cm2) as compared to the MDCK- MDR1 cells (>250 Ω∙cm2) and displayed lower Papp,a-b values. The efflux ratios obtained with iP-gp and MDCK-MDR1 monolayers correlated with Kp,uu,brain values from the in vivo studies, where compounds with the lowest Kp,uu,brain generally displayed the highest efflux ratios. 12 of the tested compounds displayed a poor BBB penetration in mice as judged by Kp,uu less than 1. Of these compounds, nine compounds were categorized as P-gp substrates in the iP-gp screening, whereas analysis of data estimated in MDCK-MDR1 cells indicated four compounds as potential substrates. The results suggest that the iP-gp cell model may be a sensitive and useful screening tool for drug screening purposes to identify possible substrates of human P-glycoprotein.

Crystalline sponge affinity screening: A fast tool for soaking condition optimization without the need of X-ray diffraction analysis.

Structural elucidation of small molecules only available in low quantity (nanogram) is one of the big advantages of the crystalline sponge method. The optimization of various soaking parameters is crucial for effective analyte absorption and repetitive positioning in the pores of the crystal. Time-consuming X-ray diffraction measurements are necessary for data collection and confirmation of successful guest inclusion. In this work, we report a screening method to select optimal soaking conditions without the need of single-crystal X-ray diffraction analysis for individual compounds and mixtures. 14 substances were chosen as test compounds. Parallel guest soaking of individual compounds and mixtures was conducted using various soaking conditions. After evaporation of solvent, excessive material was removed, and guest molecules released through dissolution of the framework. Liquid chromatography-tandem mass spectrometry allowed the estimation of analyte trapped in the pores and the selection of optimal soaking condition dependent on the highest amount of analyte to crystal size (affinity factor). The tool allowed subsequent crystallographic analysis of ten compounds with minimal experiment time. Additionally, a study to examine the lower limit of detection of the crystalline sponge method was conducted. Determination of two target analytes was possible using only 5 ng of sample. Our study shows the potential of an affinity screening to prioritize soaking parameters by estimation of the guest concentration in a single crystal for one or multiple target compounds within a short period of time.

Design and Synthesis of Pyrrolo[2,3-d]pyrimidine-Derived Leucine-Rich Repeat Kinase 2 (LRRK2) Inhibitors Using a Checkpoint Kinase 1 (CHK1)-Derived Crystallographic Surrogate.

Inhibitors of leucine-rich repeat kinase 2 (LRRK2) and mutants, such as G2019S, have potential utility in Parkinson's disease treatment. Fragment hit-derived pyrrolo[2,3-d]pyrimidines underwent optimization using X-ray structures of LRRK2 kinase domain surrogates, based on checkpoint kinase 1 (CHK1) and a CHK1 10-point mutant. (2R)-2-Methylpyrrolidin-1-yl derivative 18 (LRRK2 G2019S cKi 0.7 nM, LE 0.66) was identified, with increased potency consistent with an X-ray structure of 18/CHK1 10-pt. mutant showing the 2-methyl substituent proximal to Ala147 (Ala2016 in LRRK2). Further structure-guided elaboration of 18 gave the 2-[(1,3-dimethyl-1H-pyrazol-4-yl)amino] derivative 32. Optimization of 32 afforded diastereomeric oxolan-3-yl derivatives 44 and 45, which demonstrated a favorable in vitro PK profile, although they displayed species disconnects in the in vivo PK profile, and a propensity for P-gp- and/or BCRP-mediated efflux in a mouse model. Compounds 44 and 45 demonstrated high potency and exquisite selectivity for LRRK2 and utility as chemical probes for the study of LRRK2 inhibition.

Synthesis and Characterization of the Novel Rodent-Active and CNS-Penetrant P2X7 Receptor Antagonist Lu AF27139.

There remains an insufficient number of P2X7 receptor antagonists with adequate rodent potency, CNS permeability, and pharmacokinetic properties from which to evaluate CNS disease hypotheses preclinically. Herein, we describe the molecular pharmacology, safety, pharmacokinetics, and functional CNS target engagement of Lu AF27139, a novel rodent-active and CNS-penetrant P2X7 receptor antagonist. Lu AF27139 is highly selective and potent against rat, mouse, and human forms of the receptors. The rat pharmacokinetic profile is favorable with high oral bioavailability, modest clearance (0.79 L/(h kg)), and good CNS permeability. In vivo mouse CNS microdialysis studies of lipopolysaccharide (LPS)-primed and 2'(3')-O-(benzoylbenzoyl)adenosine-5'-triphosphate (BzATP)-induced IL-1ß release demonstrate functional CNS target engagement. Importantly, Lu AF27139 was without effect in standard in vitro and in vivo toxicity studies. Based on these properties, we believe Lu AF27139 will be a valuable tool for probing the role of the P2X7 receptor in rodent models of CNS diseases.