Multidrug-resistant Escherichia coli is a continuously growing worldwide public health problem, in which the well-known AcrAB-TolC tripartite RND efflux pump is a critical driver. We have previously described pyridylpiperazines as a novel class of allosteric inhibitors of E. coli AcrB which bind to a unique site in the protein transmembrane domain, allowing for the potentiation of antibiotic activity. Here, we show a rational optimization of pyridylpiperazines by modifying three specific derivatization points of the pyridine core to improve the potency and the pharmacokinetic properties of this chemical series. In particular, this work found that the introduction of a primary amine to the pyridine through ester (29, BDM91270) or oxadiazole (44, BDM91514) based linkers allowed for analogues with improved antibiotic boosting potency through AcrB inhibition. In vitro studies, using genetically engineered mutants, showed that this improvement in potency is mediated through novel interactions with distal acidic residues of the AcrB binding pocket. Of the two leads, compound 44 was found to have favorable physico-chemical properties and suitable plasma and microsomal stability. Together, this work expands the current structure-activity relationship data on pyridylpiperazine efflux pump inhibitors, and provides a promising step towards future in vivo proof of concept of pyridylpiperazines as antibiotic potentiators.

Antimicrobial resistance (AMR) is a major public health issue, causing 5 million deaths per year. Without any action plan, AMR will be in a near future the leading cause of death ahead of cancer. AMR comes from the ability of bacteria to rapidly develop and share resistance mechanisms towards current antibiotics, rendering them less effective. To circumvent this issue and avoid the phenomenon of cross-resistance, new antibiotics acting on novel targets or with new modes of action are required. Today, the pipeline of potential new treatments with these characteristics includes promising compounds such as gepotidacin, zoliflodacin, ibezapolstat, MGB-BP-3, CRS-3123, afabicin and TXA-709, which are currently in clinical trials, and lefamulin, which has been recently approved by FDA and EMA. In this review, we report the chemical synthesis, mode of action, structure-activity relationships, in vitro and in vivo activities as well as clinical data of these eight small molecules listed above.

It is critical that novel classes of antituberculosis drugs are developed to combat the increasing burden of infections by multidrug-resistant strains. To identify such a novel class of antibiotics, a chemical library of unique 3-D bioinspired molecules was explored revealing a promising, mycobacterium specific Tricyclic SpiroLactam (TriSLa) hit. Chemical optimization of the TriSLa scaffold delivered potent analogues with nanomolar activity against replicating and nonreplicating Mycobacterium tuberculosis. Characterization of isolated TriSLa-resistant mutants, and biochemical studies, found TriSLas to act as allosteric inhibitors of type II NADH dehydrogenases (Ndh-2 of the electron transport chain), resulting in an increase in bacterial NADH/NAD+ ratios and decreased ATP levels. TriSLas are chemically distinct from other inhibitors of Ndh-2 but share a dependence for fatty acids for activity. Finally, in vivo proof-of-concept studies showed TriSLas to protect zebrafish larvae from Mycobacterium marinum infection, suggesting a vulnerability of Ndh-2 inhibition in mycobacterial infections.

Osteoarthritis (OA) is a multifactorial pathology and comprises a wide range of distinct phenotypes. In this context, the characterization of the different molecular profiles associated with each phenotype can improve the classification of OA. In particular, OA can coexist with type 2 diabetes mellitus (T2DM). This study investigates lipidomic and proteomic differences between human OA/T2DM– and OA/T2DM+ cartilage through a multimodal mass spectrometry approach. Human cartilage samples were obtained after total knee replacement from OA/T2DM– and OA/T2DM+ patients. Label-free proteomics was employed to study differences in protein abundance and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) for spatially resolved-lipid analysis. Label-free proteomic analysis showed differences between OA/T2DM– and OA/T2DM+ phenotypes in several metabolic pathways such as lipid regulation. Interestingly, phospholipase A2 protein was found increased within the OA/T2DM+ cohort. In addition, MALDI-MSI experiments revealed that phosphatidylcholine and sphingomyelin species were characteristic of the OA/T2DM– group, whereas lysolipids were more characteristic of the OA/T2DM+ phenotype. The data also pointed out differences in phospholipid content between superficial and deep layers of the cartilage. Our study shows distinctively different lipid and protein profiles between OA/T2DM– and OA/T2DM+ human cartilage, demonstrating the importance of subclassification of the OA disease for better personalized treatments.

Endoplasmic reticulum aminopeptidase 2 (ERAP2) is a key enzyme involved in the trimming of antigenic peptides presented by Major Histocompatibility Complex class I. It is a target of growing interest for the treatment of autoimmune diseases and in cancer immunotherapy. However, the discovery of potent and selective ERAP2 inhibitors is highly challenging. Herein, we have used kinetic target-guided synthesis (KTGS) to identify such inhibitors. Co-crystallization experiments revealed the binding mode of three different inhibitors with increasing potency and selectivity over related enzymes. Selected analogues engage ERAP2 in cells and inhibit antigen presentation in a cellular context. 4 d (BDM88951) displays favorable in vitro ADME properties and in vivo exposure. In summary, KTGS allowed the discovery of the first nanomolar and selective highly promising ERAP2 inhibitors that pave the way of the exploration of the biological roles of this enzyme and provide lead compounds for drug discovery efforts.

The restrictions posed by the COVID-19 pandemic obliged the French Society for Medicinal Chemistry (Société de chimie thérapeutique) and the French Microbiology Society (Société Française de Microbiologie) to organize their joint autumn symposium (entitled “On the hunt for next-generation antimicrobial agents”) online on 9–10 December 2021. The meeting attracted more than 200 researchers from France and abroad with interests in drug discovery, antimicrobial resistance, medicinal chemistry, and related disciplines. This review summarizes the 13 invited keynote lectures. The symposium generated high-level scientific dialogue on the most recent advances in combating antimicrobial resistance. The University of Lille, the Institut Pasteur de Lille, the journal Pharmaceuticals, Oxeltis, and INCATE, sponsored the event.

Introduction In addition to the well-known cartilage extracellular matrix-related expression of Sox9, we demonstrated that chondrogenic differentiation of progenitor cells is driven by a sharply defined bi-phasic expression of Sox9: an immediate early and a late (extracellular matrix associated) phase expression. In this study, we aimed to determine what biological processes are driven by Sox9 during this early phase of chondrogenic differentiation. Materials Sox9 expression in ATDC5 cells was knocked down by siRNA transfection at the day before chondrogenic differentiation or at day 6 of differentiation. Samples were harvested at 2 h and 7 days of differentiation. The transcriptomes (RNA-seq approach) and proteomes (Label-free proteomics approach) were compared using pathway and network analyses. Total protein translational capacity was evaluated with the SuNSET assay, active ribosomes were evaluated with polysome profiling, and ribosome modus was evaluated with bicistronic reporter assays. Results Early Sox9 knockdown severely inhibited chondrogenic differentiation weeks later. Sox9 expression during the immediate early phase of ATDC5 chondrogenic differentiation regulated the expression of ribosome biogenesis factors and ribosomal protein subunits. This was accompanied by decreased translational capacity following Sox9 knockdown, and this correlated to lower amounts of active mono- and polysomes. Moreover, cap- versus IRES-mediated translation was altered by Sox9 knockdown. Sox9 overexpression was able to induce reciprocal effects to the Sox9 knockdown. Conclusion Here, we identified an essential new function for Sox9 during early chondrogenic differentiation. A role for Sox9 in regulation of ribosome amount, activity, and/or composition may be crucial in preparation for the demanding proliferative phase and subsequent cartilage extracellular matrix production of chondroprogenitors in the growth plate in vivo.

Purpose: Early detection of knee osteoarthritis (OA) is critical for possible preventive treatment. Specific biomarkers for prognosis and early diagnosis of OA are lacking. Therefore, in this study, we analyzed the lipid profiles of different tissue types within Hoffa's fat pad (HFP) of OA and cartilage defect (CD) patients, using matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI). The HFP has already been shown to play an important role in the inflammatory process in OA by prostaglandin release. Additionally, MALDI-MSI allows us to investigate tissue lipid distribution at molecular level, which makes it a promising tool for the detection of disease specific factors for OA development. In this study, we demonstrate that HFP of patients with OA can be discriminated from patients with CD by identifying their lipid profiles. Methods: HFP of patients with OA (n=3) and patients with CD (n=3) were washed in Phosphate Buffered Saline (PBS) and snap frozen directly after surgical dissection. Snap frozen HFPs were cryosectioned at 15 μm (Leica Microsystems Cryotome, Wetzlar) and mounted onto indium tin oxide coated glass slides. Samples were sublimed (HTX Sublimator, HTX Imaging, Chapel Hill) with Norharmane matrix and analyzed by MALDI-MSI in positive and negative ion modes at a lateral resolution of 50 μm on a RapifleX Tissue Typer (Bruker Daltonics, Bremen). Data analysis was performed using FlexImaging 5.0 (Bruker Daltonics, Bremen) and SCiLS Lab (SCiLS, Bremen) software. Different tissue types (fat, connective tissue and synovium) within HFP were annotated after Haematoxylin & Eosin staining using QuPath version 0.2.0-m5 (GitHub, San Francisco). MS/MS was performed on a Q-Exactive (Thermo Fisher Scientific, Waltham) in consecutive slides to identify lipid species. LipidMaps and Lipostar were used for lipid assignments. Results: Multivariate analysis such as principal component analysis and discriminant analysis revealed a differential lipid profile in HFP of patients with OA (blue) or CD patients (red) in positive (A) and negative (B) ion-mode (Figure 1). Phosphatidylinositol, sphingomyelin and phosphatidylcholine related species presented different abundances. In particular, phosphatidylinositol species were associated to OA patients. Co-registration of MALDI-MSI data with histology showed specific lipid species associated to adipose tissue or connective tissue within HFP (Figure 2). This fact suggests the importance of tissue intra-heterogeneity in HFP.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Conclusions: To our knowledge, this is the first study comparing lipid profiles in HFP of OA and CD patients using MALDI-MSI. Our results show different lipid profiles between OA and CD patients, as well as intra-tissue heterogeneity within HFP, rendering MALDI-MSI as a useful technology for OA biomarker discovery. Future research will focus on expanding the number of subjects and the improvement of lipid detection signals.

The incidence of osteoarthritis (OA) is increasing in our younger population. OA development early in life is often related to cartilage damage, caused by (sport) injury or trauma. Detection of ear...

Osteochondrosis is a developmental orthopedic disease affecting growing cartilage in young horses. In this study we compared the proteomes of equine chondrocytes obtained from healthy and osteochondrotic cartilage using a label-free mass spectrometry approach. Quantitative changes of some proteins selected for their involvement in different functional pathways highlighted by the bioinformatics analysis, were validated by western blotting, while biochemical alterations of extracellular matrix were confirmed via Raman spectroscopy analysis. In total 1637 proteins were identified, of which 59 were differentially abundant. Overall, the results highlighted differentially represented proteins involved in metabolic and functional pathways that may be related to the failure of the endochondral ossification process occurring in osteochondrosis. In particular, we identified proteins involved in extracellular matrix degradation and organization, vitamin metabolism, osteoblast differentiation, apoptosis, protein folding and localization, signalling and gene expression modulation and lysosomal activities. These results provide valuable new insights to elucidate the underlying molecular mechanisms associated with the development and progression of osteochondrosis. SIGNIFICANCE: Osteochondrosis is a common articular disorder in young horses mainly due to defects in endochondral ossification. The pathogenesis of osteochondrosis is still poorly understood and only a limited number of proteomic studies have been conducted. This study provides a comprehensive characterization of proteomic alterations occurring in equine osteochondrotic chondrocytes, the only resident cell type that modulates differentiation and maturation of articular cartilage. The results evidenced alterations in abundance of proteins involved in functional and metabolic pathways and in extracellular matrix remodelling. These findings could help clarify some molecular aspects of osteochondrosis and open new fields of research for elucidating the pathogenesis of this disease.

Background: Osteoarthritis (OA) is mainly characterized by the progressive deterioration of articular cartilage. Recent studies support that type 2 diabetes (TD2) is a risk factor to develop OA [1, 2]. However, the molecular cartilage profile of patients combining these two diseases remains unclear, and a better understanding of the different OA phenotypes should be considered for the development of personalized medicine. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is used to investigate the bimolecular distribution of proteins, lipids or metabolites through the in-situ analysis of tissue sections. Bottom-up proteomics focuses on the relative quantification of proteins. The combination of both technologies could be considered to reveal specific molecular profiles and help for patient classification. Objectives: The main goal of this study is to apply a multimodal mass spectrometry approach on cartilage to reveal specific lipidomic and proteomic profiles associated to TD2 patients. Methods: Human cartilages from OA (n a =10) and OA/TD2 human patients (n b =10) were obtained from donors undergoing total knee joint replacement. Cartilage punches of 8*8mm were sectioned at 12 µm thickness for MALDI-MSI and bottom-up proteomics. For MALDI-MSI experiments (n a =6; n b =6), norharmane matrix was sprayed over the samples for the detection of lipids. Experiments were then performed in positive ion polarity at 50 µm of lateral resolution using a RapifleX MALDI Tissue-typer instrument. LipostarMSI and in-house ChemomeTricks toolbox for MATLAB software were used for data processing and analysis. For bottom-up proteomics experiment (n a =10; n b =10), proteins were extracted, separated using SDS-PAGE and digested prior to liquid chromatography separation coupled to an orbitrap MS Q-Exactive HF mass spectrometer. Proteome Discoverer, enrichR and Reactome software were used for data processing and analysis. Results: MALDI-MSI showed overall differences between OA and OA/TD2 patients based on their specific lipidomic profiles. In particular, sphingomyelin and phosphatidylcholine species were significantly more abundant in OA patients whereas lysolipids such as lysophosphatidylcholine species were mainly present in OA/TD2 patients, providing therefore phenotype-specific OA molecular panels. Additionally, we observed that phosphatidylcholine and sphingomyelin species were more present in the superficial layer of the cartilage whereas lysophosphatidylcholine species were more abundant in the deep layer (Fig. 1A, B). Proteomics experiments applied on cartilage enables the quantification of 114 proteins. Among those, 73 were overexpressed in OA samples whereas 41 were overexpressed in OA/TD2 patients. Among the differentially regulated proteins (Fig. 1C), phospholipase A2 was increased in the diabetic cohort, in line with the elevated level of lysolipids found in the imaging data. Our results also involved the fatty acid omega oxidation and the fatty acid biosynthesis pathways as relevant to explain this deregulation of the lipid metabolism. Conclusion: MALDI–MSI combined with proteomics experiments showed different profiles between OA and OA/TD2 patients and could be employed for patient classification. References: [1]Louati, K., et al., Association between diabetes mellitus and osteoarthritis: systematic literature review and meta-analysis. RMD Open, 2015. 1 (1): p. e000077. [2]Williams, M.F., et al., Type 2 diabetes and osteoarthritis: a systematic review and meta-analysis. J Diabetes Complications, 2016. 30 (5): p. 944-50. Acknowledgments: The Dutch Province of Limburg and MUMC institutional grant. Disclosure of Interests: Maxime Eveque: None declared, Pieter Emans Shareholder of: Shareholder and cofounder start-up company Chondropeptix, Grant/research support from: Institution received grants from STW, ReumaNederland, InSciTE, Consultant of: Consultancy to Kiomed, Speakers bureau: Payment for lectures by Kiomed, Episurf, Britt Claes: None declared, Freek Bouwman: None declared, Ron M A Heeren: None declared, Berta Cillero-Pastor: None declared

Purpose: Triamcinolone acetonide (TAA) is one of the drug treatments employed to ameliorate inflammation and pain characteristic of osteoarthritis (OA). However, the penetration, distribution, and the effects of this drug after intra articular injection into the avascular cartilage are not well understood yet. It is necessary to elucidate their penetrance into tissue and their concentration in the cartilage. In addition to this, the literature is still unclear on the ideal dose to treat patients. To address these issues, we used mass spectrometry imaging (MSI) to study and elucidate the penetration of TAA in human OA cartilage. In this work, we also investigated the drug effects at the lipid and protein level using mass spectrometry. Methods: Cartilage material was obtained from 5 human OA patients after full knee replacement and was incubated with TAA at different concentrations (control; 10−3M; 10−4M; 10−5M; cartilage chips at 10x10 mm). Thin tissue sections were mounted on target slides before coating them with specific matrices for matrix assisted laser desorption ionization (MALDI). MALDI-MSI was used to investigate the drug effects at the lipid level at 100 μm of spatial resolution in positive and negative ion modes whereas liquid chromatography coupled to label free MS was employed to identify protein/peptide changes. To overcome the TAA hydrophobicity, Girard T was employed to visualize the drug distribution in cartilage tissue by MSI. MALDI-2 was employed as an alternative strategy to visualize TAA distribution without the need for chemical reaction. Multivariate analysis such as principal component analysis and discriminant analysis were employed for data reduction and to look for lipid profiles characteristic of each TAA condition. Results: Our study reported for the first time that TAA derivatization combined with MALDI-MSI is an innovative way to follow the penetrance of corticosteroids within cartilage (Fig. 1a). TAA quantification in three different human OA patients showed that the concentration was patient dependent (Fig. 1b and c). Therefore, we observed that the first patient accumulated 22.18 ng/μl (± 2.11) while the concentration was only 6.80 (± 0.05) and 10.34 ng/μl (± 1.00) for the other two respectively (Fig. 1d). Long sample preparation procedures for derivatization can be avoided using the alternative MALDI-2 approach eliminating the need for an on-tissue chemical approach. In addition, we observed that the concentration of the drug indeed correlated with changes in lipid and protein profiles. After principal component analysis and discriminant analysis on MALDI-MSI data, an increase in sphingomyelin species and a decrease of lipid fragmentation was observed after 48 h of incubation with high doses of TAA. These results showed us that the tissues incubated with the higher dose of drug were close to the OA biomolecular profile indicating that 10−3M treatment had no positive effect. At the protein level, we found out that some proteins such as anti-regenerative protein CILP1 (cartilage intermediate layer protein 1) were downregulated, whereas some proteins such as collagen alpha-2(V) chain were upregulated. More interestingly, we noticed that some proteins were dose-dependent (stromelysin-1, cartilage oligomeric matrix protein). Our results show that proteins are differently regulated by the drug concentration and that lower doses seemed to be beneficial for OA tissues (Fig. 2). Conclusions: By employing MSI we have shown that TAA is able to penetrate the human OA cartilage and reach all cartilage layers. The drug accumulation observed was tissue/patient dependent. In addition to this, lipidomic and proteomic changes have been observed showing that low or medium TAA concentrations could be more beneficial for the OA pathology.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Background Osteoarthritis (OA) is one of the most common diseases, caused by a chronic degenerative disorder of the joint. OA can be related to the metabolic syndrome or metabolic abnormalities being recently defined as a subtype of the disease1. Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) technology allows for the investigation of the bimolecular distribution of proteins, lipids or metabolites through the in situ analysis of tissue sections. In order to better understand the metabolic OA phenotype, the study of the endogenous metabolic profiles using MALDI-IMS should be considered. Objectives The main goal of this study is to apply MALDI-IMS methodology to study the metabolic spatial distribution of cartilage and to reveal intra-tissue and inter-patient heterogeneity. Methods Human OA cartilage was obtained from donors undergoing total knee joint replacement. Samples were heat stabilized by a stabilizer system, before being snap frozen. Cartilage punches were sectioned at 12 μm thickness in a cryostat and deposited on indium tin oxide (ITO) glass slides. 9-Aminoacridine (9AA) and N-(1-Naphthyl) Ethylenediamine DihydroChloride (NEDC) matrices were sprayed on the tissues. MALDI-MS profiling and imaging experiments were performed using different mass spectrometers. Data were analyzed by different software dedicated to mass spectrometry. Results Results showed that 9AA and NEDC matrices were both able to extract several and different compounds. MALDI-MS/MS was employed with 9AA matrix for molecular identification, confirming for the first time the presence of several metabolites in cartilage such as adenosine triphosphate, adenosine diphosphate, uridine triphosphate or N-Acetylglucosamine. Punches from lesioned and non-lesioned areas from the same OA patient were heat stabilized and sprayed with NEDC matrix. MALDI-IMS experiments at 40-μm of spatial resolution showed a different metabolic distribution between deep and superficial areas but also between lesioned and non-lesioned regions suggesting an evidence in the existence of intra-tissue heterogeneity (figure 1). Conclusions MALDI-IMS methodology is a useful technique for metabolite profiling of cartilage and could be employed to study OA patient heterogeneity. This fact will be especially relevant for OA patients suffering of metabolic syndrome. References Zhuo, Q., et al. (2012). Metabolic syndrome meets osteoarthritis. Nat Rev Rheumatol 8(12): 729–737. Disclosure of Interest None declared