Staphylococcus aureus subcapsular splenic abscess and associated empyema in the setting of tocilizumab therapy: A case report.

We report a case of Staphylococcus aureus subcapsular splenic abscess and associated empyema after recent commencement of tocilizumab, masquerading as musculoskeletal pain. This highlights the importance of considering unusual underlying infections in patients on tocilizumab.

Discovery of DNL343: A Potent, Selective, and Brain-Penetrant eIF2B Activator Designed for the Treatment of Neurodegenerative Diseases.

Eukaryotic translation initiation factor 2B (eIF2B) is a key component of the integrated stress response (ISR), which regulates protein synthesis and stress granule formation in response to cellular insult. Modulation of the ISR has been proposed as a therapeutic strategy for treatment of neurodegenerative diseases such as vanishing white matter (VWM) disease and amyotrophic lateral sclerosis (ALS) based on its ability to improve cellular homeostasis and prevent neuronal degeneration. Herein, we report the small-molecule discovery campaign that identified potent, selective, and CNS-penetrant eIF2B activators using both structure- and ligand-based drug design. These discovery efforts culminated in the identification of DNL343, which demonstrated a desirable preclinical drug profile, including a long half-life and high oral bioavailability across preclinical species. DNL343 was progressed into clinical studies and is currently undergoing evaluation in late-stage clinical trials for ALS.

DRESS syndrome with multiorgan involvement and HHV-6 reactivation in the absence of a drug trigger.

Drug reaction with eosinophilia and systemic symptoms (DRESS) is a severe drug reaction where patients present with fever, morbilliform rash and multiorgan manifestations, which may include acute renal failure, acute respiratory distress syndrome and eosinophilic myocarditis. We present a case of a 60-year-old woman with acute heart failure, DRESS syndrome features and human herpesvirus 6 reactivation in the absence of a drug trigger. She was diagnosed with eosinophilic myocarditis and successfully treated with corticosteroid therapy.

Cutaneous leukocytoclastic vasculitis associated with verapamil and atorvastatin: A case report.

We report a case of biopsy-proven cutaneous leukocytoclastic vasculitis developing 10 days after starting verapamil and atorvastatin in a patient with long-standing Sjogren's syndrome. This highlights the need to monitor for this rare adverse effect.

Discovery of Potent and Selective Dual Leucine Zipper Kinase/Leucine Zipper-Bearing Kinase Inhibitors with Neuroprotective Properties in In Vitro and In Vivo Models of Amyotrophic Lateral Sclerosis.

Dual leucine zipper kinase (DLK) and leucine zipper-bearing kinase (LZK) are regulators of neuronal degeneration and axon growth. Therefore, there is a considerable interest in developing DLK/LZK inhibitors for neurodegenerative diseases. Herein, we use ligand- and structure-based drug design approaches for identifying novel amino-pyrazine inhibitors of DLK/LZK. DN-1289 (14), a potent and selective dual DLK/LZK inhibitor, demonstrated excellent in vivo plasma half-life across species and is anticipated to freely penetrate the central nervous system with no brain impairment based on in vivo rodent pharmacokinetic studies and human in vitro transporter data. Proximal target engagement and disease relevant pathway biomarkers were also favorably regulated in an in vivo model of amyotrophic lateral sclerosis.

A G358S mutation in the Plasmodium falciparum Na+ pump PfATP4 confers clinically-relevant resistance to cipargamin.

Diverse compounds target the Plasmodium falciparum Na+ pump PfATP4, with cipargamin and (+)-SJ733 the most clinically-advanced. In a recent clinical trial for cipargamin, recrudescent parasites emerged, with most having a G358S mutation in PfATP4. Here, we show that PfATP4G358S parasites can withstand micromolar concentrations of cipargamin and (+)-SJ733, while remaining susceptible to antimalarials that do not target PfATP4. The G358S mutation in PfATP4, and the equivalent mutation in Toxoplasma gondii ATP4, decrease the sensitivity of ATP4 to inhibition by cipargamin and (+)-SJ733, thereby protecting parasites from disruption of Na+ regulation. The G358S mutation reduces the affinity of PfATP4 for Na+ and is associated with an increase in the parasite's resting cytosolic [Na+]. However, no defect in parasite growth or transmissibility is observed. Our findings suggest that PfATP4 inhibitors in clinical development should be tested against PfATP4G358S parasites, and that their combination with unrelated antimalarials may mitigate against resistance development.

Safety, pharmacokinetics and target engagement of novel RIPK1 inhibitor SAR443060 (DNL747) for neurodegenerative disorders: Randomized, placebo-controlled, double-blind phase I/Ib studies in healthy subjects and patients.

RIPK1 is a master regulator of inflammatory signaling and cell death and increased RIPK1 activity is observed in human diseases, including Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS). RIPK1 inhibition has been shown to protect against cell death in a range of preclinical cellular and animal models of diseases. SAR443060 (previously DNL747) is a selective, orally bioavailable, central nervous system (CNS)-penetrant, small-molecule, reversible inhibitor of RIPK1. In three early-stage clinical trials in healthy subjects and patients with AD or ALS (NCT03757325 and NCT03757351), SAR443060 distributed into the cerebrospinal fluid (CSF) after oral administration and demonstrated robust peripheral target engagement as measured by a reduction in phosphorylation of RIPK1 at serine 166 (pRIPK1) in human peripheral blood mononuclear cells compared to baseline. RIPK1 inhibition was generally safe and well-tolerated in healthy volunteers and patients with AD or ALS. Taken together, the distribution into the CSF after oral administration, the peripheral proof-of-mechanism, and the safety profile of RIPK1 inhibition to date, suggest that therapeutic modulation of RIPK1 in the CNS is possible, conferring potential therapeutic promise for AD and ALS, as well as other neurodegenerative conditions. However, SAR443060 development was discontinued due to long-term nonclinical toxicology findings, although these nonclinical toxicology signals were not observed in the short duration dosing in any of the three early-stage clinical trials. The dose-limiting toxicities observed for SAR443060 preclinically have not been reported for other RIPK1-inhibitors, suggesting that these toxicities are compound-specific (related to SAR443060) rather than RIPK1 pathway-specific.

Projected Dose Optimization of Amino- and Hydroxypyrrolidine Purine PI3Kδ Immunomodulators.

The approvals of idelalisib and duvelisib have validated PI3Kδ inhibitors for the treatment for hematological malignancies driven by the PI3K/AKT pathway. Our program led to the identification of structurally distinct heterocycloalkyl purine inhibitors with excellent isoform and kinome selectivity; however, they had high projected human doses. Improved ligand contacts gave potency enhancements, while replacement of metabolic liabilities led to extended half-lives in preclinical species, affording PI3Kδ inhibitors with low once-daily predicted human doses. Treatment of C57BL/6-Foxp3-GDL reporter mice with 30 and 100 mg/kg/day of 3c (MSD-496486311) led to a 70% reduction in Foxp3-expressing regulatory T cells as observed through bioluminescence imaging with luciferin, consistent with the role of PI3K/AKT signaling in Treg cell proliferation. As a model for allergic rhinitis and asthma, treatment of ovalbumin-challenged Brown Norway rats with 0.3 to 30 mg/kg/day of 3c gave a dose-dependent reduction in pulmonary bronchoalveolar lavage inflammation eosinophil cell count.

Discovery of a new series of PI3K-δ inhibitors from Virtual Screening.

PI3K-δ mediates key immune cell signaling pathways and is a target of interest for treatment of oncological and immunological disorders. Here we describe the discovery and optimization of a novel series of PI3K-δ selective inhibitors. We first identified hits containing an isoindolinone scaffold using a combined ligand- and receptor-based virtual screening workflow, and then improved potency and selectivity guided by structural data and modeling. Careful optimization of molecular properties led to compounds with improved permeability and pharmacokinetic profile, and high potency in a whole blood assay.

A key cytosolic iron-sulfur cluster synthesis protein localizes to the mitochondrion of Toxoplasma gondii.

Iron-sulfur (Fe-S) clusters are prosthetic groups on proteins that function in a range of enzymatic and electron transfer reactions. Fe-S cluster synthesis is essential for the survival of all eukaryotes. Independent Fe-S cluster biosynthesis pathways occur in the mitochondrion, plastid, and cytosolic compartments of eukaryotic cells. Little is known about the cytosolic Fe-S cluster biosynthesis in apicomplexan parasites, the causative agents of diseases such as malaria and toxoplasmosis. NBP35 serves as a key scaffold protein on which cytosolic Fe-S clusters assemble, and has a cytosolic localization in most eukaryotes studied thus far. Unexpectedly, we found that the NBP35 homolog of the apicomplexan Toxoplasma gondii (TgNBP35) localizes to the outer mitochondrial membrane, with mitochondrial targeting mediated by an N-terminal transmembrane domain. We demonstrate that TgNBP35 is critical for parasite proliferation, but that, despite its mitochondrial localization, it is not required for Fe-S cluster synthesis in the mitochondrion. Instead, we establish that TgNBP35 is important for the biogenesis of cytosolic Fe-S proteins. Our data are consistent with TgNBP35 playing a central and specific role in cytosolic Fe-S cluster biosynthesis, and imply that the assembly of cytosolic Fe-S clusters occurs on the cytosolic face of the outer mitochondrial membrane in these parasites.

Fragment-based lead discovery of a novel class of small molecule antagonists of neuropeptide B/W receptor subtype 1 (GPR7).

Here, we report the discovery of a new class of NPBWR1 antagonists identified from a fragment-based screen. Compound 1 (cAMP IC50 = 250 µM; LE = 0.29) emerged as an initial hit. Further optimization of 1 by SAR-by-catalogue and chemical modification produced 21a (cAMP IC50 = 30 nM; LE = 0.39) with a 6700-fold increase in potency from fragment 1. Somewhat surprisingly, Schild analysis of compound 21a suggested that in vitro inhibition of NPW-mediated effects on upon cAMP accumulation were saturable, and that compound 21a dose-dependently increased [125I]-hNPW23 dissociation rate constants from NPBWR1 in kinetic binding studies. Collectively, these data are inconsistent with a classic surmountable, orthosteric mechanism of inhibition. The benzimidazole inhibitors reported herein may therefore represent a mechanistically differentiated class of compounds with which to form a better appreciation of the pharmacology and physiological roles of this central neuropeptide system.

Detecting changes at the leading edge of an interface between oceanic water layers.

Many physical phenomena in the ocean involve interactions between water masses of different temperatures and salinities at boundaries. Of particular interest is the characterisation of finescale structure at the marginal interaction zones of these boundaries, where the structure is either destroyed by mixing or formed by stratification. Using high-resolution seismic reflection imaging, we present observations of temporal changes at the leading edge of an interface between sub-thermocline layers in the Panama Basin. By studying time-lapse images of a seismic reflector between two water boundaries with subtle differences, we provide empirical constraints on how stratified layers evolve. The leading edge of this reflector, which is characterised by a gradual lateral decrease in vertical temperature contrast ([Formula: see text]), increases in length over ~3 days coupled with an increase in [Formula: see text]. A critical mixing state, in which turbulent diffusion is gradually replaced by double-diffusion as the dominant mixing process, is thus revealed.

Improvement of hERG-ROMK index of spirocyclic ROMK inhibitors through scaffold optimization and incorporation of novel pharmacophores.

SAR in the previously described spirocyclic ROMK inhibitor series was further evolved from lead 4 by modification of the spirocyclic core and identification of novel right-side pharmacophores. In this process, it was discovered that the spiropyrrolidinone core with the carbonyl group α to the spirocenter was preferred for potent ROMK activity. Efforts aimed at decreasing hERG affinity within the series led to the discovery of multiple novel right-hand pharmacophores including 3-methoxythiadiazole, 2-methoxypyrimidine, and pyridazinone. The most promising candidate is pyridazinone analog 32 that showed an improved functional hERG/ROMK potency ratio and preclinical PK profile. In vivo evaluation of 32 demonstrated blood pressure lowering effects in the spontaneously hypertensive rat model.

The design and synthesis of novel spirocyclic heterocyclic sulfone ROMK inhibitors as diuretics.

A spirocyclic class of ROMK inhibitors was developed containing a structurally diverse heterocyclic sulfone moiety and spirocyclic core starting from lead 1. These compounds not only displayed exquisite ROMK potency but significantly improved selectivity over hERG. The lead compounds were found to have favorable pharmacokinetic properties and displayed robust diuretic, natriuretic and blood pressure lowering effects in spontaneously hypertensive rats.

Discovery of MK-7145, an Oral Small Molecule ROMK Inhibitor for the Treatment of Hypertension and Heart Failure.

ROMK, the renal outer medullary potassium channel, is involved in potassium recycling at the thick ascending loop of Henle and potassium secretion at the cortical collecting duct in the kidney nephron. Because of this dual site of action, selective inhibitors of ROMK are expected to represent a new class of diuretics/natriuretics with superior efficacy and reduced urinary loss of potassium compared to standard-of-care loop and thiazide diuretics. Following our earlier work, this communication will detail subsequent medicinal chemistry endeavors to further improve lead selectivity against the hERG channel and preclinical pharmacokinetic properties. Pharmacological assessment of highlighted inhibitors will be described, including pharmacodynamic studies in both an acute rat diuresis/natriuresis model and a subchronic blood pressure model in spontaneous hypertensive rats. These proof-of-biology studies established for the first time that the human and rodent genetics accurately predict the in vivo pharmacology of ROMK inhibitors and supported identification of the first small molecule ROMK inhibitor clinical candidate, MK-7145.

Discovery of a Potent and Selective ROMK Inhibitor with Pharmacokinetic Properties Suitable for Preclinical Evaluation.

A new subseries of ROMK inhibitors exemplified by 28 has been developed from the initial screening hit 1. The excellent selectivity for ROMK inhibition over related ion channels and pharmacokinetic properties across preclinical species support further preclinical evaluation of 28 as a new mechanism diuretic. Robust pharmacodynamic effects in both SD rats and dogs have been demonstrated.

Discovery of benzimidazole oxazolidinediones as novel and selective nonsteroidal mineralocorticoid receptor antagonists.

Elaboration of the oxazolidinedione series led to replacement of the exocyclic amides with substituted benzimidazoles. The structure-activity relationship (SAR) exploration resulted in the discovery of potent and selective nonsteroidal mineralocorticoid receptor (MR) antagonists with significantly improved microsomal stability and pharmacokinetic (PK) profile relative to the HTS hit 1a. One compound 2p possessed comparable efficacy as spironolactone (SPL) at 100 mg/kg (p.o.) in the rat natriuresis model. As such, this series was validated as a lead series for further optimization.

Metabolic activation and major protein target of a 1-benzyl-3-carboxyazetidine sphingosine-1-phosphate-1 receptor agonist.

1-{4-[(4-Phenyl-5-trifluoromethyl-2-thienyl)methoxy]benzyl}azetidine-3-carboxylic acid (MRL-A) is a potent sphingosine-1-phosphate-1 receptor agonist, with potential application as an immunosuppressant in organ transplantation or for the treatment of autoimmune diseases. When administered orally to rats, radiolabeled MRL-A was found to undergo metabolism to several reactive intermediates, and in this study, we have investigated its potential for protein modification in vivo and in vitro. MRL-A irreversibly modified liver and kidney proteins in vivo, in a dose- and time-dependent manner. The binding was found to occur selectively to microsomal and mitochondrial subcellular fractions. Following a nonspecific proteolytic digestion of liver and kidney proteins, a single major amino acid adduct was observed. This adduct was characterized with LC/MS/UV and NMR spectroscopy and was found to be the product of an unprecedented metabolic activation of the azetidine moiety leading to the formation of a ring-opened α,ß-unsaturated imine conjugated to the ε-amino group of a lysine residue. The formation of this adduct was not inhibited when rats were pretreated with 1-aminobenzotriazole, indicating that P450 enzymes were not involved in the metabolic activation of MRL-A. Rather, our findings suggested that MRL-A underwent bioactivation via a ß-oxidation pathway. Several other minor adducts were identified from protein hydrolysates and included lysine, serine, and cysteine conjugates of MRL-A. These minor adducts were also detected in microsomal incubations fortified with the cofactors for acyl-CoA synthesis and in hepatocytes. Trypsin digestion of crude liver homogenates from rats treated with radiolabeled MRL-A led to the identification of a single radioactive peptide. Its sequence, determined by LC/MS analysis, revealed that the target of the major reactive species of MRL-A in vivo is Lys676 of long chain acyl-CoA synthetase-1 (ACSL1). This lysine residue has been found to be critical for ACSL1 activity, and its modification has the potential to lead to biological consequences such as cardiac hypertrophy or thermogenesis dysregulation.

The discovery of potent antagonists of NPBWR1 (GPR7).

The synthesis and evaluation of small molecule antagonists of the G protein-coupled receptor NPBWR1 (GPR7) are reported for the first time. [4-(5-Chloropyridin-2-yl)piperazin-1-yl][(1S,2S,4R)-4-{[(1R)-1-(4-methoxyphenyl)ethyl]amino}-2-(thiophen-3-yl)cyclohexyl]methanone (1) emerged as a hit from a high-throughput screen. Examination of substituents that focused on replacing the 5-chloropyridine and 4-methoxybenzylamino groups of 1 led to the identification of compounds that exhibited subnanomolar potencies as low as 660pM (9k) in the functional assay and 200pM in the binding assay (9i).

Discovery of a highly potent, selective, and bioavailable soluble epoxide hydrolase inhibitor with excellent ex vivo target engagement.

4-Substituted piperidine-derived trisubstituted ureas are reported as highly potent and selective inhibitors for sEH. The SAR outlines approaches to improve activity against sEH and reduce ion channel and CYP liability. With minimal off-target activity and a good PK profile, the benchmark 2d exhibited remarkable in vitro and ex vivo target engagement. The eutomer entA-2d also elicited vasodilation effect in rat mesenteric artery.