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NOD2 (nucleotide-binding oligomerization domain-containing protein 2) is an internal pattern recognition receptor that recognizes bacterial peptidoglycan and stimulates host immune responses. Dysfunction of NOD2 pathway has been associated with a number of autoinflammatory disorders. To date, direct inhibitors of NOD2 have not been described due to technical challenges of targeting the oligomeric protein complex. Receptor interacting protein kinase 2 (RIPK2) is an intracellular serine/threonine/tyrosine kinase, a key signaling partner, and an obligate kinase for NOD2. As such, RIPK2 represents an attractive target to probe the pathological roles of NOD2 pathway. To search for selective RIPK2 inhibitors, we employed virtual library screening (VLS) and structure based design that eventually led to a potent and selective RIPK2 inhibitor 8 with excellent oral bioavailability, which was used to evaluate the effects of inhibition of RIPK2 in various in vitro assays and ex vivo and in vivo pharmacodynamic models.

In vitro absorption, distribution, metabolism and elimination (ADME) assays are widely used for profiling compounds in pharmaceutical drug discovery programs. Many compounds are screened in metabolic stability assays, using liver microsomes as a model of intrinsic hepatic clearance. Analysis of metabolic stability assays has relied on high throughput LC-MS/MS techniques to keep up with automated assays and compound profiling needs. An experimental alternative to sample analysis via fast chromatography employs an open port interface (OPI) which dilutes and directs acoustically-ejected droplets from microtiter plates to a conventional electrospray ion source for ionization and introduction into a mass spectrometer. Metabolic stability assays of 37 commercial drug compounds using in human, dog, rat and mouse liver microsomes (LMs), were analyzed by LC-MS/MS and an experimental breadboard version of an ADE-OPI-MS/MS system. Results from the experiments comparing intrinsic clearance (CLint) generated with ADE-OPI-MS/MS vs fast LC-MS/MS for all compounds showed ≥86% of CLint values were within a factor of two with R2 ≥ 0.86 using 25 nL and 5 nL sample ejection volumes on the ADE-OPI-MS/MS instrument. Throughput with the experimental ADE-OPI-MS/MS system used in this study was more than ten-fold faster than analysis by the fast LC-MS/MS at 1.3 s/sample versus 17.2 s/sample, respectively.

Imidazopyridine 1 was identified from a phenotypic screen against P. falciparum (Pf) blood stages and subsequently optimized for activity on liver-stage schizonts of the rodent parasite P. yoelii (Py) as well as hypnozoites of the simian parasite P. cynomolgi (Pc). We applied these various assays to the cell-based lead optimization of the imidazopyrazines, exemplified by 3 (KAI407), and show that optimized compounds within the series with improved pharmacokinetic properties achieve causal prophylactic activity in vivo and may have the potential to target the dormant stages of P. vivax malaria.

On the basis of the initial success of optimization of a novel series of imidazolopiperazines, a second generation of compounds involving changes in the core piperazine ring was synthesized to improve antimalarial properties. These changes were carried out to further improve the potency and metabolic stability of the compounds by leveraging the outcome of a set of in vitro metabolic identification studies. The optimized 8,8-dimethyl imidazolopiperazine analogues exhibited improved potency, in vitro metabolic stability profile and, as a result, enhanced oral exposure in vivo in mice. The optimized compounds were found to be more efficacious than the current antimalarials in a malaria mouse model. They exhibit moderate oral exposure in rat pharmacokinetic studies to achieve sufficient multiples of the oral exposure at the efficacious dose in toxicology studies.

The synthesis and structure-activity relationship of a series of arylaminoethyl amide cathepsin S inhibitors are reported. Optimization of P3 and P2 groups to improve overall physicochemical properties resulted in significant improvements in oral bioavailability over early lead compounds. An X-ray structure of compound 37 bound to the active site of cathepsin S is also reported.

Nanoelectrospray ionization (nanoESI) mass spectrometry was performed on naturally occurring steroid sulfates and unconjugated steroids derivatized to their sulfate esters using precursor ion monitoring. Initially, an extraction method was developed based on a combinatorial approach employed to obtain the most efficient liquid/liquid extraction protocol. The new method allowed unconjugated steroids and their sulfated analogues to be isolated separately in a two-step procedure using diethyl ether/hexane (90:10, v/v) in the first step to extract the unconjugated steroids and chloroform/2-butanol (50:50, v/v) in the second step to extract steroid sulfates. Precursor ion scanning performed with a triple-quadrupole mass spectrometer was used to examine quantitatively the extracted unconjugated and sulfated steroids, where the recovery efficiency averaged 70 and 87%, respectively. In addition, some steroids could be structurally elucidated by employing tandem mass spectrometry. The limit of detection for steroid sulfates from the biological matrix was 200 amol/microL (approximately 80 fg/microL) with only 1 microL of sample being injected. Endogenous levels of the unconjugated and sulfated steroids were detected and quantified from physiological samples including urine and blood. Internal standards, pregnenolone-d4 sulfate and dehydroepiandrosterone-d2 (DHEA), were used for quantitation. Extraction and nanoESI analyses were also performed on cerebrospinal fluid where the neurosteroid DHEA sulfate was detected. The small amount of material consumed (typically less than 20% of the injection volume) suggests that nanoESI has even greater potential for high sensitivity when combined with nanoLC approaches, especially for monitoring reproductive and adrenal steroids, as well as for the analysis of the less abundant neurosteroids.

[Image: see text] Starting from a hit series from a GNF compound library collection and based on a cell-based proliferation assay of Plasmodium falciparum, a novel imidazolopiperazine scaffold was optimized. SAR for this series of compounds is discussed, focusing on optimization of cellular potency against wild-type and drug resistant parasites and improvement of physiochemical and pharmacokinetic properties. The lead compounds in this series showed good potencies in vitro and decent oral exposure levels in vivo. In a Plasmodium berghei mouse infection model, one lead compound lowered the parasitemia level by 99.4% after administration of 100 mg/kg single oral dose and prolonged mice survival by an average of 17.0 days. The lead compounds were also well-tolerated in the preliminary in vitro toxicity studies and represents an interesting lead for drug development.

Pharmacologic antagonism of cannabinoid 1 receptors (CB1 receptors) in the central nervous system (CNS) suppresses food intake, promotes weight loss, and improves the metabolic profile. Since the CB1 receptor is expressed both in the CNS and in peripheral tissues, therapeutic value may be gained with CB1 receptor inverse agonists acting on receptors in both domains. The present report examines the metabolic and CNS actions of a novel CB1 receptor inverse agonist, compound 64, a 1,5,6-trisubstituted pyrazolopyrimidinone. Compound 64 showed similar or superior binding affinity, in vitro potency, and pharmacokinetic profile compared to rimonabant. Both compounds improved the metabolic profile in diet-induced obese (DIO) rats and obese cynomolgus monkeys. Weight loss tended to be greater in compound 64-treated DIO rats compared to pair-fed counterparts, suggesting that compound 64 may have metabolic effects beyond those elicited by weight loss alone. In the CNS, reversal of agonist-induced hypothermia and hypolocomotion indicated that compound 64 possessed an antagonist activity in vivo. Dosed alone, compound 64 suppressed extinction of conditioned freezing (10 mg/kg) and rapid eye movement (REM) sleep (30 mg/kg), consistent with previous reports for rimonabant, although for REM sleep, compound 64 was greater than threefold less potent than for metabolic effects. Together, these data suggested that (1) impairment of extinction learning and REM sleep suppression are classic, centrally mediated responses to CB1 receptor inverse agonists, and (2) some separation may be achievable between central and peripheral effects with brain-penetrating CB1 receptor inverse agonists while maintaining metabolic efficacy. Furthermore, chronic treatment with compound 64 contributes to evidence that peripheral CB1 receptor blockade may yield beneficial outcomes that exceed those elicited by weight loss alone.

Peptidic, non-covalent inhibitors of lysosomal cysteine protease cathepsin S (1 and 2) were investigated due to low oral bioavailability, leading to an improved series of peptidomimetic inhibitors. Utilizing phenyl succinamides as the P2 residue increased the oral exposure of this lead series of compounds, while retaining selective inhibition of the cathepsin S isoform. Concurrent investigation of the P1 and P2 subsites resulted in the discovery of several potent and selective inhibitors of cathepsin S with good pharmacokinetic properties due to the elimination of saturated aliphatic P2 residues.

A systematic study of anilines led to the discovery of a metabolically robust fluoroindoline replacement for the alkoxy aniline toxicophore in 1. Investigations of the P1 pocket resulted in the discovery of a wide tolerance of functionality leading to the discovery of 11 as a potent and selective inhibitor of cathepsin S.

Electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry have been used to examine evidence in a sexual assault investigation. Because condoms are being used increasingly by sexual assailants and some condom brands include the spermicide nonoxynol-9 (nonylphenoxy polyethoxyethanol) in the lubricant formulation, the recovery, and identification of nonoxynol-9 from evidence items may assist in proving corpus delicti. A method was developed for the recovery of nonoxynol-9 from internal vaginal swabs and for its identification by reverse phase liquid chromatography/electrospray ionization mass spectrometry (LC ESI-MS), nanoelectrospray ionization (nanoESI) mass spectrometry, and high resolution MALDI Fourier transform mass spectrometry (MALDI-FTMS). The method was tested on extracts from precoitus, immediate postcoitus, and four-hours postcoitus vaginal swabs provided by a volunteer whose partner does not normally use condoms, but for this trial used a condom having a water-soluble gel-type lubricant that includes 5% nonoxynol-9 in its formulation. Subsequently, LC ESI-MS was used to identify traces of nonoxynol-9 from the internal vaginal swab of a victim of a sexual assault.

Pharmacokinetics is the study of the absorption, distribution, metabolism, and excretion (ADME) of drugs; the respective rates of which are of fundamental importance in determining their effect on the organism to which they are administered. Pharmacokinetic studies with mass spectrometry can provide quantitative information about a compound’s half-life in the body and how quickly it is metabolized or excreted. These studies are also used to determine drug distribution, partitioning within an organism, and drug dosing regime. The latter is important in determining a drug’s therapeutic range, which is a balance between the concentration required for positive therapeutic effect versus higher doses which may have toxicological effects. Finally, pharmacokinetics is used to investigate drug dosing with regards to administration factors of age, gender, ethnicity, concomitant drugs, or diseases. Pharmacokinetic analysis involves the determination of the concentration of drugs and/or their metabolites in biological fluids, which may be present at very low concentrations. Quantitative analysis is important since a major consideration in these types of studies is to determine how much drug gets into the systemic circulation. The integration of drug concentration over time (the so-called Area-Under-TheCurve or AUC) is a measure of both how well the drug is cleared by an individual and how well the analytical system is performing. Therefore, reliable, sensitive and specific methods are required for these types of analyses. With the increased pressure on researchers and pharmaceutical companies to discover and develop novel therapeutic compounds in a rapid manner, mass spectrometry now plays a central role in pharmacokinetics studies. The initial mass spectrometric technique used in pharmacokinetics, gas chromatography/mass spectrometry (GC/MS) was limited to molecules that were either volatile or could be made thermally stable by pre-column derivatization. Derivatization is often used to improve a molecule’s ability to be vaporized and ionized with a GC/MS, yet it has significant disadvantages because many molecules are still not amenable to GC/MS analysis after derivatization and the sample handling associated with derivatization results in sample loss. More recently, liquid chromatography coupled with soft ionization techniques such as electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI) with triple quadrupole mass analysis (Fig. 1), have become the most common approaches in pharmacokinetic studies. This is because