Novel bacterial topoisomerase inhibitors (NBTIs) target bacterial topoisomerases through binding modes distinct from fluoroquinolones but are often limited by hERG channel inhibition. Our previous findings indicated that the introduction of a non-aminopiperidine linker and a halogenated phenyl enzyme-binding moiety can partially reduce cardiotoxicity. In this paper, we describe whether optimizing the DNA-intercalating 1,5-naphthyridine moiety could further improve safety while maintaining antibacterial potency. Several optimized compounds showed strong enzyme inhibition and potent antibacterial activity, particularly against Gram-positive pathogens. Compound 13 displayed broad-spectrum activity, with MICs as low as 0.008 μg/mL against Gram-positive and 0.125 μg/mL against Gram-negative bacteria. Safety profiling revealed reduced cytotoxicity and hERG binding compared to earlier series. Compounds 6, 20, 24, and 25 were nontoxic in zebrafish embryo assays. Compound 13 showed toxicity only at high doses and protected embryos in a lethalStaphylococcus aureus infection model, highlighting its potential as a preclinical lead.

Antibiotic-resistant Staphylococcus aureus poses a major global health challenge as current therapies lose effectiveness. Because most S. aureus infections are biofilm-associated, there is a critical need for new agents capable of targeting biofilm-embedded cells, and not just their planktonic form. Novel bacterial type II topoisomerase inhibitors (NBTIs) are known for their excellent antibacterial properties, against planktonic bacteria, and could also possess anti-biofilm activity. To investigate this, we designed and synthesized a new series of NBTIs featuring alternative linker scaffolds and a modified DNA-intercalating moiety to improve antibacterial and antibiofilm activity. Comprehensive MIC profiling against clinically relevant Gram-negative and Gram-positive bacteria revealed clear structure-activity trends, with compounds 20 and 40 emerging as the most promising candidates. Compound 20 showed exceptional potency against S. aureus and multiple MRSA strains (MICs 0.004-0.008 μg/mL), while compound 40 displayed the most balanced broad-spectrum activity. Both compounds exhibited dual inhibition of DNA gyrase and topoisomerase IV, maintained high selectivity over human topoisomerase IIα. In vivo evaluation in zebrafish embryos demonstrated robust protection against lethal S. aureus infection within a well-tolerated concentration range. Kinetic studies demonstrated dose-dependent bactericidal activity and measurable post-antibiotic effects. Additionally, both compounds inhibited biofilm formation at 1/2 MIC and reduced metabolic activity within mature biofilms at 2 × MIC, outperforming gepotidacin, an FDA-approved NBTI, in several assays. Together, these findings identify new NBTI chemotypes with enhanced potency and promising antibiofilm activity, providing a strong foundation for future antibacterial development.

Radiolabeled gastrin antagonists are emerging as a new tool for the diagnosis and treatment of cholecystokinin-2 receptor (CCK2R) expressing tumors such as medullary thyroid carcinoma and small cell lung cancer, overcoming limitations of agonist-based ligands. In this study, the CCK2R antagonist nastorazepide (Z-360) was used as a targeting scaffold in combination with either a peptidic (NPP) or a hydrophilic polysaccharide (NPS) linker and the chelators DOTA and DOTAGA, yielding four novel radioligands. These were evaluated alongside the reference minigastrin analogs DOTA-PP-F11 and DOTA-MGS5. Methods:All ligands were synthetized via solid-phase peptide synthesis and labeled with In-111 and Lu-177. In vitro characterization included receptor affinity, hydrophilicity and cellular uptake in A431-CCK2R. In vivo biodistribution and imaging were performed in A431-CCK2R xenografts. [177Lu]Lu-DOTAGA-NPP was further assessed in a proof-of-concept therapy study and metabolic stability assays. Results:111In-labeled antagonists showed 3-4-fold higher site recognition than [111In]In-DOTA-PP-F11 and nanomolar affinities comparable to the reference ligand. Among the tested ligands, [111In]In-DOTAGA-NPP demonstrated the highest cellular uptake (22.0 ± 0.2%), approximately twice that of the other ligands and moderately higher than [111In]In-DOTA-PP-F11 (16.2 ± 1.8%) after 4 h at 37 °C. In vivo, all 177Lu-labeled antagonists demonstrated high tumor uptake (range: 22.98 ± 7.04 - 29.00 ± 3.30% I.A./g tissue) with [177Lu]Lu-DOTAGA-NPP exhibiting the most favorable tumor-to-tissue ratios. Compared with [177Lu]Lu-DOTA-MGS5, [177Lu]Lu-DOTAGA-NPP showed rapid tumor accumulation with faster washout but reduced stomach exposure and high in vivo stability. Conclusion:[¹⁷⁷Lu]Lu-DOTAGA-NPP emerged as the most promising lead compound, combining high tumor uptake, favorable tumor-to-organ ratios, and excellent in vivo stability. Although no significant therapeutic efficacy was observed in the tested xenograft model, the results support [177Lu]Lu-DOTAGA-NPP as a promising lead for optimization. Overall, these findings highlight the potential of Z-360-based CCK2R antagonists as scaffolds for next-generation theranostic radioligands and support their development as alternatives to minigastrin-based tracers.

N-glycosylation is a key post-translational modification of proteins influencing their physicochemical properties as well as biological activity. This study investigates the in vivo activity of four β-1,4-galactosyltransferase 1 inhibitors in cell cultures producing recombinant IgG antibodies. Inhibitors, previously identified as potent inhibitors in on-target assay, were evaluated in fed-batch bioprocesses. Dose-dependent effects on cell culture performance were assessed following compound addition. N-glycan profiles were analyzed using InstantPC labeling and UHPLC-FLD, while intracellular compound concentrations were quantified via LC-MS/MS following time-resolved sampling of treated cell lysates. Bioprocess parameters, including cell growth, viability, and productivity, were monitored throughout cultivation. The study revealed divergent behaviors between on-target potency and in vivo activity, underscoring the importance of cellular permeability, metabolic stability, and off-target effects of potential inhibitors used in cell culture. These findings provide new insights into the challenges of glycosylation pathway modulation and inform strategies for future glycoengineering tool development.

Sialyltransferases catalyze the transfer of sialic acid to glycoconjugates within the Golgi apparatus, generating cell-surface sialoglycans that regulate cell-cell and immune communication. This process can be modulated by inhibiting α-2,6-sialyltransferase 1 (ST6GAL1), one of the most important sialyltransferases. Most β-galactoside ST6GAL1 inhibitors are polar CMP-Neu5Ac derivatives with limited membrane permeability, a limitation shared by the noncarbohydrate inhibitor JFD 00458. Here, we designed JFD 00458 analogs to improve permeability while maintaining ST6GAL1 inhibition and direct target engagement. Incorporation of sulfonic-acid bioisosteres, particularly sulfonylureas, improved passive permeability, as measured by parallel artificial membrane permeability assay, with the best analog reaching log(P_app [cm/s]) = -4.3. Potency was also improved in an expanded series of sulfonylureas, with the best inhibitor displaying an IC50 of 1.9 µM. Microscale thermophoresis confirmed direct ST6GAL1 binding, with similar apparent Kd values of approximately 22-25 µM for JFD 00458 and the optimized sulfonylureas. Saturation transfer difference NMR identified the substituted phenoxy phenyl core as a common ST6GAL1-contacting epitope, while closer contacts from the sulfonylurea-linked aromatic substituents in 6d and 6e were associated with the highest inhibitory potency across the series. These results establish sulfonylureas as membrane-permeable sulfonic-acid bioisosteres and provide a structure-activity framework for further ST6GAL1 inhibitor optimization.

Seven different enzymes comprise the galactosyltransferases family, of which beta-1,4-galactosyltransferase I (beta-1,4-GALT1) is the major contributor to galactosylation activity in cells. Since abnormalities in galactosylation are associated with many pathophysiological conditions, beta-1,4-GALT1 is an interesting new target for drug discovery and molecular probe design. There are several known beta-1,4-GALT1 inhibitors, but most of them suffer from low cell permeability and thus low in vivo activity. In the present work, we describe an in silico screening performed using commercially available virtual compound libraries that led us to the discovery of novel beta-1,4-GALT1 inhibitors. A virtual screening campaign was performed by docking compound libraries to the binding site of beta-1,4-GALT1, followed by biological evaluation of selected hits for their beta-1,4-GALT1 inhibitory activity. The IC50 values were determined for the best performing inhibitors to obtain new chemotypes of beta-1,4-GALT1 inhibitors.

In the present work, we describe the design, synthesis, and evaluation of a galectin-8-binding fluorescent probe designed for a competitive fluorescence polarization (FP) assay for screening new galectin-8N inhibitors. The probe was characterized for its photophysical properties and its binding affinity for galectin-8N was determined by using FP. We evaluated the probe in a competitive FP assay with three known galectin-8N inhibitors and demonstrated its suitability for high-throughput screening.

We report here the use of Tris-BODIPY-OH as a scaffold for the multivalent display of sugar heads. A chloroacetyl thioether ligation reaction easily yields mannosylated BODIPYs, named Man9-BODIPY and (Man-TEG)9-BODIPY, which display nine mannose residues. Regardless of the linker length, both glycoBODIPYs provide an arrangement of mannose heads that allows for proper recognition by the carbohydrate binding domain of concanavalin A (ConA). Moreover, the interactions of Man9-BODIPY with relevant human lectins, i.e. dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) and langerin, were further investigated. The approach proposed is versatile and paves the way for the development of multivalent and fluorescent glyco-BODIPY probes useful to interrogate carbohydrate-lectin interactions in different biological contexts.

Galectin-8 is a tandem-repeat galectin consisting of two distinct carbohydrate recognition domains and is a potential drug target. We have developed a library of galectin-8N inhibitors that exhibit high nanomolar Kd values as determined by a competitive fluorescence polarization assay. A detailed thermodynamic analysis of the binding of D-galactosides to galectin-8N by isothermal titration calorimetry reveals important differences in enthalpic and/or entropic contributions to binding. Contrary to expectations, the binding of 2-O-propargyl-D-galactoside was found to strongly increase the binding enthalpy, whereas the binding of 2-O-carboxymethylene-D-galactoside was surprisingly less enthalpy-driven. The results of our work suggest that the ethynyl group can successfully replace the carboxylate group when targeting the water-exposed guanidine moiety of a critical arginine residue. This results in only a minor loss of affinity and an adjusted enthalpic contribution to the overall binding due to non-canonical cation-π interactions, as evidenced by the obtained crystal structure of 2-O-propargyl-D-galactoside in complex with the N-terminal domain of galectin-8. Such an interaction has neither been identified nor discussed to date in a small-molecule ligand-protein complex.

Cardio-cerebrovascular diseases are increasingly prevalent worldwide, with pathological changes in the heart and brain reinforcing each other. Diabetes is a major driver of comorbidity between these two systems and contributes to poor clinical outcomes. This review summarizes current evidence on shared risk factors and underlying mechanisms, with a particular focus on the role of dipeptidyl peptidase-4 (DPP-4) inhibitors as a potential therapeutic strategy for preserving cardio-cerebrovascular homeostasis. Growing evidence suggests that DPP-4 inhibitors offer benefits beyond glycemic control. These include improvements in endothelial function, reduction of oxidative stress and inflammatory responses, modulation of lipid and glucose metabolism, and regulation of blood pressure. Together, these actions support the anti-atherosclerotic and anti-thromboembolic properties of this drug class. These effects occur through both indirect pathways, via improved glycemic control, and direct cellular and molecular mechanisms. Although DPP-4 inhibitors are commonly used as second- or third-line agents in combination with other antidiabetic drugs, they have distinct advantages in specific populations. Notably, they are among the few hypoglycemic agents that are safe and effective in patients with impaired renal function—where the use of agents like metformin and SGLT-2 inhibitors is often contraindicated. In addition, DPP-4 inhibitors have shown favorable outcomes in elderly patients, particularly those aged 65 years and older. From a cardiovascular perspective, DPP-4 inhibitors have demonstrated protective effects against ischemic stroke, improved neurovascular function, and a reduction in major adverse cardiovascular events (MACEs). Importantly, they do not increase the risk of heart failure, unlike some other antidiabetic medications. While most cardiovascular outcome trials (CVOTs) involving DPP-4 inhibitors have shown neutral results, these studies were primarily designed to establish safety rather than demonstrate cardiovascular superiority. In conclusion, the pleiotropic effects, favorable safety profile, and suitability for vulnerable populations position DPP-4 inhibitors as promising agents in the management of cardio-cerebrovascular complications in diabetes. Further long-term, controlled clinical studies are warranted to fully establish their therapeutic potential across broader indications.

The O-GlcNAc transferase (OGT) inhibitors disclosed thus far are burdened with certain shortcomings. We have incorporated OSMI-4, a nanomolar OGT inhibitor, into PROTACs. The prepared chimeras failed to induce effective OGT degradation or influence O-GlcNAcylation levels in MM.1S cells.

Antimicrobial resistance caused by the excessive and inappropriate use of antibacterial drugs is a global health concern. Currently, we are walking a fine line between the fact that most bacterial infections can still be cured with the antibiotics known so far, and the emergence of infections with bacteria resistant to several drugs at the same time, against which we no longer have an effective drug. Therefore, new antibacterial drugs are urgently needed to curb the hard-to-treat infections. Our group has developed new antibacterials from the class of novel bacterial topoisomerase inhibitors (NBTIs) that exhibit broad-spectrum antibacterial activity. This article reviews our efforts in developing highly potent NBTIs over the past decade. Following the discovery of an initial hit with potent enzyme inhibitory and broad-spectrum antibacterial activity, an extensive hit-to-lead campaign was conducted with the goal of optimizing physicochemical properties, reducing hERG inhibition, and maintaining antibacterial activity against both Gram-positive and Gram-negative bacteria, with a focus on methicillin-resistant Staphylococcus aureus (MRSA). This optimization strategy resulted in an amide-containing, focused NBTI library with compounds exhibiting potent antibacterial activity against Gram-positive bacteria, reduced hERG inhibition, no cardiotoxicity in in vivo zebrafish model, and favorable in vivo efficacy in a neutropenic murine thigh infection model for MRSA infections.

Glycosylation of recombinant proteins is a post-translational modification that affects multiple physicochemical and biological properties of proteins. As such, it is a critical quality attribute that must be carefully controlled during protein production in the pharmaceutical industry. Glycosylation can be modulated by various conditions, including the composition of production media and feeds. In this study, the N-glycosylation-modulating effects of numerous compounds, including metal enzyme cofactors, enzyme inhibitors, and metabolic intermediates, were evaluated. Chinese hamster ovary cells producing three different IgG antibodies were cultivated in a fed-batch mode. First, a one-factor-at-a-time experiment was performed in 24-well deep well plates to identify the strongest modulators and appropriate concentration ranges. Then, a full response surface experiment was designed to gauge the effects and interactions of the 14 most effective hit compounds in an Ambr® 15 bioreactor system. A wide range of glycoform content was achieved, with an up to eight-fold increase in individual glycoforms compared to controls. The resulting model can be used to determine modulator combinations that will yield desired glycoforms in the final product.

In this Letter, we present a small series of novel bacterial topoisomerase inhibitors (NTBIs) that exhibit both potent inhibition of Mycobacterium tuberculosis DNA gyrase and potent antimycobacterial activity. The disclosed crystal structure of M. tuberculosis DNA gyrase in complex with DNA and compound 5 from this NBTI series reveals the binding mode of an NBTI in the GyrA binding pocket and confirms the presence and importance of halogen bonding for the excellent on-target potency. In addition, we have shown that compound 5 is a promising M. tuberculosis DNA gyrase inhibitor, with an IC50 for M. tuberculosis gyrase of 0.096 mu M, and it has potent activity against M. tuberculosis, with an IC50 of 0.165 mu M.

Bacteria are capable of remarkable adaptations to their environment, including undesirable bacterial resistance to antibacterial agents. One of the most serious cases is an infection caused by multidrug-resistant Staphylococcus aureus, which has unfortunately also spread outside hospitals. Therefore, the development of new effective antibacterial agents is extremely important to solve the increasing problem of bacterial resistance. The bacteriolytic enzyme autolysin E (AtlE) is a promising new drug target as it plays a key role in the degradation of peptidoglycan in the bacterial cell wall. Consequently, disruption of function can have an immense impact on bacterial growth and survival. An in silico and in vitro evaluation of iminosugar derivatives as potent inhibitors of S. aureus (AtlE) was performed. Three promising hit compounds (1, 3 and 8) were identified as AtlE binders in the micromolar range as measured by surface plasmon resonance. The most potent compound among the SPR response curve hits was 1, with a KD of 19 μM. The KD value for compound 8 was 88 μM, while compound 3 had a KD value of 410 μM.

Galectins are among organisms' most abundantly expressed lectins (carbohydrate-binding proteins) that specifically bind β-galactosides. They act not only outside the cell, where they bind to extracellular matrix glycans, but also inside the cell, where they have a significant impact on signaling pathways. Galectin-8 is a galectin family protein encoded by the LGALS8 gene. Its role is evident in both T- and B-cell immunity and in the innate immune response, where it acts directly on dendritic cells and induces some pro-inflammatory cytokines. Galectin-8 also plays an important role in the defense against bacterial and viral infections. It is known to promote antibacterial autophagy by recognizing and binding glycans present on the vacuolar membrane, thus acting as a danger receptor. The most important role of galectin-8 is the regulation of cancer growth, metastasis, tumor progression, and tumor cell survival. Importantly, the expression of galectins is typically higher in tumor tissues than in noncancerous tissues. In this review article, we focus on galectin-8 and its function in immune response, microbial infections, and cancer. Given all of these functions of galectin-8, we emphasize the importance of developing new and selective galectin-8 inhibitors and report the current status of their development.

Galectin-8 contains two different carbohydrate recognition domains (CRDs). Selective inhibitors for at least one CRD are desirable for galectin-8 biology studies and potentially for pharmacological purposes. Structure-guided design led to the discovery of potent and selective glycomimetic-heterocycle hybrid ligands, with a 4-(p-bromophenyl)phthalazinone derivative displaying a 34 mu M K-d for galectin-8N (N-terminal CRD), no binding to galectin-8C (C-terminal CRD), -1, -3, -4N, -7, -9C, or -9N, and >40-fold selectivity over galectin-4C. Selectivity was achieved with the halogenated 4-phenylphthalazinone moiety occupying a galectin-8N-specific sub-pocket. A 1.30 & Aring; resolution X-ray structure revealed the phthalazinone moiety stacking with Arg45 and the 4-bromophenyl moiety stacking both Arg59 and Tyr141 of galectin-8N. Physicochemical and in vitro ADME studies revealed a desirable LogD, which also translated to good passive permeability. The chemical, microsome, and plasma stability support these compounds as promising tool compounds and candidates for hit-to-lead optimization.

Background: The novel bacterial topoisomerase inhibitors (NBTIs) developed in our laboratory show potent on-target enzyme inhibition but suffer from low activity against Gram-negative bacteria. Methods: With the aim of improving the antibacterial activity of our compounds against Gram-negative bacteria, we tested them in combination with different efflux pump inhibitors (EPIs), a strategy that showed promise in several other classes of antimicrobials. We also investigated the combined effect of NBTIs with ATP-competitive inhibitors of bacterial type II topoisomerases (ACIs), as well as the antibiofilm properties of our compounds and the combination with EPIs against early and mature Acietobacter baumannii biofilm. Results: Our results demonstrate that combinations of NBTIs with EPI Phenylalanine-arginyl-β-naphthylamide significantly reduce the corresponding NBTIs’ minimal inhibitory concentration values and show potentiation of A. baumannii biofilm inhibition as compared to NBTIs alone. Although combinations of NBITs and ACIs did not show synergistic effects, the FIC index value calculations revealed additive effects for all the combinations of a selected NBTI in combination with three ACIs in all the assayed Gram-negative bacteria from the ESKAPE group. Conclusions: These results show for the first time that combinations of NBTIs with either EPIs or a different class of the topoisomerase inhibitors may be a beneficial strategy to combat difficult-to-treat bacterial infections.

Finding potent inhibitors of O-GlcNAc transferase (OGT) has proven to be a challenge, especially because the diversity of published inhibitors is low. The large majority of available OGT inhibitors are uridine-based or uridine-like compounds that mimic the main interactions of glycosyl donor UDP-GlcNAc with the enzyme. Until recently, screening of DNA-encoded libraries for discovering hits against protein targets was dedicated to a few laboratories around the world, but has become accessible to wider public with the recent launch of the DELopen platform. Here we report the results and follow-up of a DNA-encoded library screening by using the DELopen platform. This led to the discovery of two new hits with structural features not resembling UDP. Small focused libraries bearing those two scaffolds were made, leading to low micromolar inhibition of OGT and elucidation of their structure-activity relationship

β‐ N ‐Acetylglucosamine transferase (OGT) inhibition is considered an important topic in medicinal chemistry. The involvement of O‐GlcNAcylation in several important biological pathways is pointing to OGT as a potential therapeutic target. The field of OGT inhibitors drastically changed after the discovery of the 7‐quinolone‐4‐carboxamide scaffold and its optimization to the first nanomolar OGT inhibitor: OSMI‐4. While OSMI‐4 is still the most potent inhibitor reported to date, its physicochemical properties are limiting its use as a potential drug candidate as well as a biological tool. In this study, we have introduced a simple modification (elongation) of the peptide part of OSMI‐4 that limits the unwanted cyclisation during OSMI‐4 synthesis while retaining OGT inhibitory potency. Secondly, we have kept this modified peptide unchanged while incorporating new sulfonamide UDP mimics to try to improve binding of newly designed OGT inhibitors in the UDP‐binding site. With the use of computational methods, a small library of OSMI‐4 derivatives was designed, prepared and evaluated that provided information about the OGT binding pocket and its specificity toward quinolone‐4‐carboxamides.