Optimization of Selectivity and Pharmacokinetic Properties of Salt-Inducible Kinase Inhibitors that Led to the Discovery of Pan-SIK Inhibitor GLPG3312.

Salt-inducible kinases (SIKs) SIK1, SIK2, and SIK3 are serine/threonine kinases and form a subfamily of the protein kinase AMP-activated protein kinase (AMPK) family. Inhibition of SIKs in stimulated innate immune cells and mouse models has been associated with a dual mechanism of action consisting of a reduction of pro-inflammatory cytokines and an increase of immunoregulatory cytokine production, suggesting a therapeutic potential for inflammatory diseases. Following a high-throughput screening campaign, subsequent hit to lead optimization through synthesis, structure-activity relationship, kinome selectivity, and pharmacokinetic investigations led to the discovery of clinical candidate GLPG3312 (compound 28), a potent and selective pan-SIK inhibitor (IC50: 2.0 nM for SIK1, 0.7 nM for SIK2, and 0.6 nM for SIK3). Characterization of the first human SIK3 crystal structure provided an understanding of the binding mode and kinome selectivity of the chemical series. GLPG3312 demonstrated both anti-inflammatory and immunoregulatory activities in vitro in human primary myeloid cells and in vivo in mouse models.

Diagnostic accuracy of a rapid nucleic acid test for group A streptococcal pharyngitis using saliva samples: protocol for a prospective multicenter study in primary care.

BACKGROUND: Group A streptococcus is found in 20-40% of cases of childhood pharyngitis; the remaining cases are viral. Streptococcal pharyngitis ("strep throat") is usually treated with antibiotics, while these are not indicated in viral cases. Most guidelines recommend relying on a diagnostic test confirming the presence of group A streptococcus before prescribing antibiotics. Conventional first-line tests are rapid antigen detection tests based on throat swabs. Recently, rapid nucleic acid tests were developed; they allow the detection of elements of the genome of group A streptococcus. We hypothesize that these rapid nucleic acid tests are sensitive enough to be performed on saliva samples instead of throat swabs, which could be more convenient in practice. METHODS: This is a multicenter, prospective diagnostic accuracy study evaluating the performance of a rapid nucleic acid test for group A streptococcus (Abbott ID NOW STREP A2) in saliva, compared with a conventional pharyngeal rapid antigen detection test (EXACTO PRO STREPTATEST, lateral flow assay, comparator test), with a composite reference standard of throat culture and group A streptococcus PCR in children with pharyngitis in primary care (i.e., 27 primary care pediatricians or general practitioners). To ensure group A streptococcus is not missed, the salivary rapid nucleic acid test requires a minimally acceptable value of sensitivity (primary outcome) set at 80%. Assuming 35% of participants will have group A streptococcus, we will recruit 800 consecutive children with pharyngitis. Secondary outcomes will include difference in sensitivity between the pharyngeal rapid antigen detection test and the salivary rapid nucleic acid test; variability in sensitivity and specificity of the salivary rapid nucleic acid test with the level of McIsaac score; time to obtain the result of the salivary rapid nucleic acid test; patient, physician, and parents satisfaction; and barriers and facilitators to using rapid tests for group A streptococcus in primary care. ETHICS AND DISSEMINATION: Approved by the Institutional Review Board "Comité de protection des personnes Ile de France I" (no. 2022-A00085-38). Results will be presented at international meetings and disseminated in peer-reviewed journals. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov: NCT05521568.

Assessment of SARS-CoV-2 testing in children during a low prevalence period (VIGIL study 1).

OBJECTIVES: SARS-CoV-2 induces a broad spectrum of clinical manifestations, which overlap with other viral infections very common in children. We aimed to describe the percentage of positive SARS-CoV-2 RT-PCR tests in symptomatic and asymptomatic ambulatory children and to determine the predictive factors for positivity. PATIENTS AND METHODS: From June 1 to July 31, 2020, we conducted a cross-sectional prospective, multicenter study (13 hospital emergency units and 59 ambulatory pediatricians) throughout France. Children under 15 years of age with a prescription of nasopharyngeal SARS-CoV-2 RT-PCR test were enrolled. RESULTS: Among the 1,553 RT-PCR tests, 22 were positive (1.4%; 95%CI [0.9; 2.1]). In both univariate and multivariate analyses, the predictive factors for positivity were age below 2 years (OR: 4.5 [1.6; 12.7]) and history of contact (OR: 12.3 [4.6; 32.8]). CONCLUSIONS: In an epidemic stage with low SARS-CoV-2 circulation, sampling of children with nonspecific symptoms and without known contact could be questioned.

Discovery of GLPG1972/S201086, a Potent, Selective, and Orally Bioavailable ADAMTS-5 Inhibitor for the Treatment of Osteoarthritis.

There are currently no approved disease-modifying osteoarthritis (OA) drugs (DMOADs). The aggrecanase ADAMTS-5 is key in the degradation of human aggrecan (AGC), a component of cartilage. Therefore, ADAMTS-5 is a promising target for the identification of DMOADs. We describe the discovery of GLPG1972/S201086, a potent and selective ADAMTS-5 inhibitor obtained by optimization of a promising hydantoin series following an HTS. Biochemical activity against rat and human ADAMTS-5 was assessed via a fluorescence-based assay. ADAMTS-5 inhibitory activity was confirmed with human aggrecan using an AGC ELISA. The most promising compounds were selected based on reduction of glycosaminoglycan release after interleukin-1 stimulation in mouse cartilage explants and led to the discovery of GLPG1972/S201086. The anticatabolic activity was confirmed in mouse cartilage explants (IC50 < 1.5 µM). The cocrystal structure of GLPG1972/S201086 with human recombinant ADAMTS-5 is discussed. GLPG1972/S201086 has been investigated in a phase 2 clinical study in patients with knee OA (NCT03595618).

2020 French recommendations on the management of septic arthritis in an adult native joint.

Septic arthritis (SA) in an adult native joint is a rare condition but a diagnostic emergency due to the morbidity and mortality and the functional risk related to structural damage. Current management varies and the recommendations available are dated. The French Rheumatology Society (SFR) Bone and Joint Infection Working Group, together with the French Language Infectious Diseases Society (SPILF) and the French Orthopaedic and Trauma Surgery Society (SOFCOT) have worked according to the HAS methodology to devise clinical practice recommendations to diagnose and treat SA in an adult native joint. One new focus is on the importance of microbiological documentation (blood cultures and joint aspiration) before starting antibiotic treatment, looking for differential diagnoses (microcrystal detection), the relevance of a joint ultrasound to guide aspiration, and the indication to perform a reference X-ray. A cardiac ultrasound is indicated only in cases of SA involving Staphylococcus aureus, oral streptococci, Streptococcus gallolyticus or Enterococcus faecalis, or when infective endocarditis is clinically suspected. Regarding treatment, we stress the importance of medical and surgical collaboration. Antibiotic therapies (drugs and durations) are presented in the form of didactic tables according to the main bacteria in question (staphylococci, streptococci and gram-negative rods). Probabilistic antibiotic therapy should only be used for patients with serious symptoms. Lastly, non-drug treatments such as joint drainage and early physical therapy are the subject of specific recommendations.

Discovery, Structure-Activity Relationship, and Binding Mode of an Imidazo[1,2-a]pyridine Series of Autotaxin Inhibitors.

Autotaxin (ATX) is a secreted enzyme playing a major role in the production of lysophosphatidic acid (LPA) in blood through hydrolysis of lysophosphatidyl choline (LPC). The ATX-LPA signaling axis arouses a high interest in the drug discovery industry as it has been implicated in several diseases including cancer, fibrotic diseases, and inflammation, among others. An imidazo[1,2-a]pyridine series of ATX inhibitors was identified out of a high-throughput screening (HTS). A cocrystal structure with one of these compounds and ATX revealed a novel binding mode with occupancy of the hydrophobic pocket and channel of ATX but no interaction with zinc ions of the catalytic site. Exploration of the structure-activity relationship led to compounds displaying high activity in biochemical and plasma assays, exemplified by compound 40. Compound 40 was also able to decrease the plasma LPA levels upon oral administration to rats.

Expression, purification and functional characterization of Wnt signaling co-receptors LRP5 and LRP6.

Activation of the Wnt signaling cascade plays a pivotal role during development and in various disease states. Wnt signals are transduced by seven-transmembrane Frizzled (Fz) proteins and the single-transmembrane LDL-receptor-related proteins 5 or 6 (LRP5/6). Genetic mutations resulting in a loss or gain of function of LRP5 in humans lead to osteopenia and bone formation, respectively. These findings demonstrate the genetic link between LRP5 signaling and the regeneration of bone mass. Herein we describe for the first time the production and characterization of soluble ectodomains of LRP5 and LRP6, (EC-LRP5, EC-LRP6). We have produced these proteins in amounts that are compatible with both in vitro and cell-based assays to study their binding properties. Purified EC-LRP5 and EC-LRP6 were able to interact with Wnt signaling components Dkk1 and Dkk2 and their functionality was confirmed in cell-based Wnt signaling assays. Hence, tools are now available to explore LRP5/6 interaction with other proteins and to screen for synthetic or natural compounds and biologics that might be novel therapeutics targeting the Wnt pathway.

Expression, purification and characterization of murine Dkk1 protein.

Dickkopf-1 (Dkk1) protein is a secreted inhibitor of canonical Wnt signaling and modulates that pathway during embryonic development. It is also implicated in several diseases and hence Dkk1 is a potential target for therapeutic intervention. In the present study 6His-tagged Dkk1 expression and secretion was assessed in five mammalian cell types. Only FreeStyle 293-F cells showed significant Dkk1 protein expression in culture medium. High and stable expression of the Dkk1 protein was obtained from a selected stable FreeStyle 293-F clone 3F8, that grows in suspension in serum-free medium. The 3F8 clone showed a high Dkk1 production level (10mg/L) for up to 2 months of culture. A one step purification procedure resulting in large amounts of highly pure and active Dkk1 protein was developed. Purified Dkk1 binds its receptors LRP5 and LRP6, and is able to dose dependently inhibit canonical Wnt signaling. Recombinant Dkk1 is glycosylated, but this modification is not essential for its biological activity. In summary, an abundant source of pure and functionally active Dkk1 protein is developed that will support the identification of inhibitors such as neutralizing antibodies that could find therapeutic use.

Expression, purification, characterization and crystallization of a recombinant human cytosolic beta-glucosidase produced in insect cells.

Human cytosolic beta-glucosidase is a monomeric enzyme that hydrolyzes various beta-d-glycosides and its real physiological role remains unclear. Here, we describe the production of this enzyme in Sf9 cells with a N-terminal 6x His tag. The production yield of the recombinant protein was in the 10 to 30 mg/l range. The protein was purified to homogeneity using two chromatographic steps, taking advantage of the 6x His tag in the first step, then using the physical and chemical properties of the protein for ionic exchange. Gel filtration analysis revealed that the protein is monomeric as expected. The kinetic parameters for 4-methylumbelliferyl beta-L-glucopyranoside, VM and KM, were measured (KM=32 microM and VM=157 micromol/h/mg at pH 7.0) and found similar to those reported for either the natural isolated enzyme or the recombinant protein expressed in COS7 cells (KM of 60-70 microM and 40 microM, respectively). Protein crystals were obtained and are now under structural investigations. In summary, we set up a heterologous expression system in Sf9 insect cells allowing the expression and production of large amounts of a pure active human protein, suitable for crystallographic studies.

MDM2 promotes proteasome-dependent ubiquitin-independent degradation of retinoblastoma protein.

Inactivation of retinoblastoma protein (Rb) plays a critical role in the development of human malignancies. It has been shown that Rb is degraded through a proteasome-dependent pathway, yet the mechanism is largely unclear. MDM2 is frequently found amplified and overexpressed in a variety of human tumors. In this study, we find that MDM2 promotes Rb degradation in a proteasome-dependent and ubiquitin-independent manner. We show that Rb, MDM2, and the C8 subunit of the 20S proteasome interact in vitro and in vivo and that MDM2 promotes Rb-C8 interaction. Expression of wild-type MDM2, but not the mutant MDM2 defective either in Rb interaction or in RING finger domain, promotes cell cycle S phase entry independent of p53. Furthermore, MDM2 ablation results in Rb accumulation and inhibition of DNA synthesis. Taken together, these findings demonstrate that MDM2 is a critical negative regulator for Rb and suggest that MDM2 overexpression contributes to cancer development by destabilizing Rb.

Epstein-Barr virus EBNA3 proteins bind to the C8/alpha7 subunit of the 20S proteasome and are degraded by 20S proteasomes in vitro, but are very stable in latently infected B cells.

A yeast two-hybrid screen using EBNA3C as bait revealed an interaction between this Epstein-Barr virus (EBV)-encoded nuclear protein and the C8 (alpha7) subunit of the human 20S proteasome. The interaction was confirmed by glutathione S-transferase (GST) pull-down experiments and these also revealed that the related proteins EBNA3A and EBNA3B can bind similarly to C8/alpha7. The interaction between these viral proteins and GST-C8/alpha7 was shown to be significantly more robust than the previously reported interaction between C8/alpha7 and the cyclin-dependent kinase inhibitor p21(WAF1/CIP1). Co-immunoprecipitation of the EBNA3 proteins with C8/alpha7 was also demonstrated after transfection of expression vectors into B cells. Consistent with this ability to bind directly to an alpha-subunit of the 20S proteasome, EBNAs 3A, 3B and 3C were all degraded in vitro by purified 20S proteasomes. However, surprisingly, no sign of proteasome-mediated turnover of these latent viral proteins in EBV-immortalized B cells could be detected, even in the presence of gamma interferon. In actively proliferating lymphoblastoid cell lines, EBNAs 3A, 3B and 3C appear to be remarkably stable, with no evidence of either de novo synthesis or proteasome-mediated degradation.

Interaction between LRP5 and Frat1 mediates the activation of the Wnt canonical pathway.

Low density lipoprotein receptor-related protein 5 (LRP5) has been identified as a Wnt co-receptor involved in the activation of the beta-catenin signaling pathway. To improve our understanding of the molecular mechanisms by which LRP5 triggers the canonical Wnt signaling cascade, we have screened for potential partners of LRP5 using the yeast two-hybrid system and identified Frat1 as a protein interacting with the cytoplasmic domain of LRP5. We demonstrate here that LRP5/Frat1 interaction is involved in beta-catenin nuclear translocation and TCF-1 transcriptional activation. The addition of Wnt3a or overexpression of constitutively active truncated LRP5 (LRP5C) induces Frat1 recruitment to the cell membrane. Overexpression of a dominant negative form of disheveled (Dvl) shows that this protein positively affects LRP5/Frat1 interaction. Furthermore, the fact that dominant negative Dvl does not interfere with LRP5C/Frat1 interaction can explain how LRP5C is capable of acting independently of this major Wnt signaling player. Axin, which has been shown to interact with LRP5 and to be recruited to the membrane through this interaction, was found to co-immunoprecipitate with Frat1 and LRP5. We propose that recruitment of Axin and Frat1 to the membrane by LRP5 leads to both Axin degradation and Frat1-mediated inhibition of glycogen synthase kinase-3. As a consequence, beta-catenin is no longer bound to Axin or phosphorylated by glycogen synthase kinase-3, resulting in TCF-1 activation.