Isothermal Titration Calorimetry Research Articles

A Complex Interplay Between Melatonin and RORβ: RORβ is Unlikely a Putative Receptor for Melatonin as Revealed by Biophysical Assays.

A nuclear retinoic acid receptor (RAR)-related orphan receptor β (RORβ) is strictly expressed in the brain, particularly in the pineal gland where melatonin is primarily synthesized and concentrated. The controversial issues regarding the direct interaction of melatonin toward ROR receptors have prompted us to investigate the potential melatonin binding sites on different ROR isoforms. We adopted computational and biophysical approaches to investigate the potential of melatonin as the ligand for RORs, in particular RORβ. Herein, possible melatonin binding sites were predicted by molecular docking on human RORs. The results showed that melatonin might be able to bind within the ligand-binding domain (LBD) of all RORs, despite their difference in sequence homology. The predicted melatonin binding scores were comparable to binding energies with respect to those of melatonin interaction to the well-characterized membrane receptors, MT1 and MT2. Although the computational analyses suggested the binding potential of melatonin to the LBD of RORβ, biophysical validation failed to confirm the binding. Melatonin was unable to alter the stability of human RORβ as shown by the unaltered melting temperatures upon melatonin administration in differential scanning fluorometry (DSF). A thermodynamic isothermal titration calorimetry (ITC) profile showed that melatonin did not interact with human RORβ in solutions, even in the presence of SRC-1 co-activator peptide. Although the direct interaction between the LBD of RORβ could not be established, RORα and RORβ gene expressions were increased upon 24h treatment with μM-range melatonin. Our data, thus, support the studies that the nuclear effects of melatonin may not be directly mediated via its interaction with the RORβ. These findings warrant further investigation on how melatonin interacts with ROR signaling and urge the melatonin research community for a paradigm shift in the direct interaction of melatonin toward RORs. The quest to identify nuclear receptors for melatonin in neuronal cells remains valid for the community to achieve.

Mixing Order Asymmetry in Nanoparticle-Polymer Complexation and Precipitation Revealed by Isothermal Titration Calorimetry.

In recent years, there has been a renewed interest in complex coacervation, driven by concerted efforts to offer novel experimental and theoretical insights into electrostatic charge-induced association. While previous studies have primarily focused on polyelectrolytes, proteins, or surfactants, our work explores the potential of using cerium (CeO2) and iron (γ-Fe2O3) oxide nanoparticles (NPs) to develop innovative nanomaterials. By combining various charged species, such as polyelectrolytes, charged neutral block copolymers, and coated NPs, we study a wide variety of complexation patterns and compare them using isothermal titration calorimetry, light scattering, and microscopy. These techniques confirm that the titration of oppositely charged species occurs in two steps: the formation of polyelectrolyte complexes and subsequent phase (or microphase) separation, depending on the system studied. Across all examined cases, the entropic contribution to the total free energy surpasses the enthalpic contribution, in agreement with counterion release mechanisms. Furthermore, our investigation reveals a consistent asymmetry in the reaction enthalpy associated with the secondary process, with exothermic profiles observed upon the addition of cationic species to anionic ones and endothermic profiles in the reverse case.

The sunflower jacalin Helja: biological and structural insights of its antifungal activity against Candida albicans.

The limited availability of efficient treatments for Candida infections and the increased emergence of antifungal-resistant strains stimulates the search for new antifungal agents. We have previously isolated a sunflower mannose-binding lectin (Helja) with antifungal activity against Candida albicans, capable of binding mannose-bearing oligosaccharides exposed on the cell surface. This work aimed to investigate the biological and biophysical basis of Helja's binding to C. albicans cell wall mannans and its influence on the fungicidal activity of the lectin. We evaluated the interaction of Helja with the cell wall mannans extracted from the isogenic parental strain (WT) and a glycosylation-defective C. albicans with altered cell wall phosphomannosylation (mnn4∆ null mutants) and investigated its antifungal effect. Helja exhibited stronger antifungal activity on the mutant strain, showing greater inhibition of fungal growth, loss of cell viability, morphological alteration, and formation of clusters with agglutinated cells. This differential biological activity of Helja was correlated with the biophysical parameters determined by solid phase assays and isothermal titration calorimetry, which demonstrated that the lectin established stronger interactions with the cell wall of the mnn4∆ null mutant than with the WT strain. In conclusion, our results provide new evidence on the nature of the Helja molecular interactions with cell wall components, i.e. phosphomannan, and its impact on the antifungal activity. This study highlights the relevance of plant lectins in the design of effective antifungal therapies.

Caffeine weakens the astringency of epigallocatechin gallate by inhibiting its interaction with salivary proteins

The astringency of green tea is an integrated result of the synergic and antagonistic effects of individual tea components, whose mechanism is highly complex and not completely understood. Herein, we used an epigallocatechin gallate (EGCG)/caffeine (CAF)/saliva model to simulate the oral conditions during tea drinking. The effect of CAF on the interaction between EGCG and salivary proteins was first investigated using molecular docking and isothermal titration calorimetry (ITC). Then, the rheological properties and the micro-network structure of saliva were studied to relate the molecular interactions and perceived astringency. The results revealed that CAF partially occupied the binding sites of EGCG to salivary proteins, inhibiting their interaction and causing changes in the elastic network structure of the salivary film, thereby reducing astringency.

The dual role of a novel Sinorhizobium meliloti chemotaxis protein CheT in signal termination and adaptation.

Sinorhizobium meliloti senses nutrients and compounds exuded from alfalfa host roots and coordinates an excitation, termination, and adaptation pathway during chemotaxis. We investigated the role of the novel S. meliloti chemotaxis protein CheT. While CheT and the Escherichia coli phosphatase CheZ share little sequence homology, CheT is predicted to possess an α-helix with a DXXXQ phosphatase motif. Phosphorylation assays demonstrated that CheT dephosphorylates the phosphate-sink response regulator, CheY1~P by enhancing its decay two-fold but does not affect the motor response regulator CheY2~P. Isothermal Titration Calorimetry (ITC) experiments revealed that CheT binds to a phosphomimic of CheY1~P with a KD of 2.9 μM, which is 25-fold stronger than its binding to CheY1. Dissimilar chemotaxis phenotypes of the ΔcheT mutant and cheT DXXXQ phosphatase mutants led to the hypothesis that CheT exerts additional function(s). A screen for potential binding partners of CheT revealed that it forms a complex with the methyltransferase CheR. ITC experiments confirmed CheT/CheR binding with a KD of 19 μM, and a SEC-MALS analysis determined a 1:1 and 2:1 CheT/CheR complex formation. Although they did not affect each other's enzymatic activity, CheT binding to CheY1~P and CheR may serve as a link between signal termination and sensory adaptation.

Preliminary exploration of the anti-ovarian cancer activity of peptides derived from bovine bone collagen hydrolysate and its related mechanisms

Ovarian cancer, a malignant tumor that poses a significant threat to women's health, has seen a rise in incidence, prompting the urgent need for more effective treatment. This study primarily aimed to explore the potential of bovine collagen peptides in inhibiting ovarian cancer. The investigation in this study began with the identification of 268 peptide sequences through LC-MS/MS, followed by a screening process using molecular docking techniques to identify potential peptides capable of binding to EGFR. Subsequently, a series of experiments were performed, demonstrating the inhibitory effects of the peptide GPAGADGDRGEAGPAGPAGPAGPR on the proliferation of ovarian cancer cells. Transcriptomic analysis further revealed that this peptide can regulate cholesterol metabolism in ovarian cancer cells. Finally, a combination of time-resolved fluorescence resonance energy transfer, isothermal titration calorimetry, molecular docking, and molecular dynamics simulations were utilized to validate the ability of this peptide to bind to the epidermal growth factor receptor (EGFR) and impede the binding of epidermal growth factor (EGF) and EGFR.

Strengthening an Intramolecular Non-Classical Hydrogen Bond to Get in Shape for Binding.

In this research article, we report on the strengthening of a non-classical hydrogen bond (C-H···O) by introducing electron withdrawing groups at the carbon atom. The approach is demonstrated on the example of derivatives of the physiological E-selectin ligand sialyl Lewisx (1, sLex). Its affinity is mainly due to a beneficial entropy term, which is predominantly caused by the pre-organization of sLex in its binding conformation. We have shown, that among the elements responsible for the pre-organization, the stabilization by a non-classical hydrogen bond between the H-C5 of l-fucose and the ring oxygen O5 of the neighboring d-galactose moiety is essential and yields 7.4 kJ mol-1. This effect could be further strengthened by replacing l-fucose by 6,6,6-trifluoro-l-fucose leading to an improved non-classical H-bond of 14.9 kJ mol-1, i.e., an improved pre-organization in the bioactive conformation. For a series of glycomimetics of sLex (1), this outcome could be confirmed by high field NMR-shifts of the H-C5Fuc, by X-ray diffraction analysis of glycomimetics co-crystallized with E-selectin as well as by isothermal titration calorimetry. Furthermore, the electron-withdrawing character of the CF3-group beneficially influences the pharmacokinetic properties of sLex mimetics. Thus, acid-stability a prerequisite for gastrointestinal stability could be substantially improved.

Modular and Photoreversible Polymer-Nanoparticle Hydrogels via Host-Guest Interactions.

Polymer-nanoparticle (PNP) hydrogels are a class of nanocomposite materials showing potential as injectable platforms for biomedical applications. Their design is limited by incomplete knowledge of how the binding motif impacts the viscoelastic properties of the material and is generally constrained to non-responsive supramolecular interactions. Expanding the scope of available interactions and advancing the understanding of how defined interactions influence network formation would accelerate PNP hydrogel design. To address this gap in the design of PNP hydrogels, the study designs and investigates a tunable platform based on beta-cyclodextrin (βCD) host-guest cross-links between functionalized polymers and nanoparticles. A host-functionalized polymer (βCD hyaluronic acid) and guest harboring block co-polymer (poly(ethylene glycol)-b-poly(lactic acid)) NPs are synthesized. The presence and accessibility for binding of the host and guest moieties are characterized via isothermal titration calorimetry. PNP hydrogels with varying concentrations of functionalized polymer and NPs reveal a limited window of concentrations for gelation. It is hypothesized that network formation is governed by the capacity of polymer chains to effectively bridge NPs, which is related to the host-guest ratios present in the system. Further, photo-responsive guests are incorporated to engineer photoreversible gelation of PNP hydrogels via exposure to specific wavelengths of light.

Supramolecular chemistry in cyclodextrin inclusion complexes: The formation rules of terpenes/β-cyclodextrin inclusion complexes

Supramolecular chemistry of cyclodextrins (CDs) inclusion complexes is a hot research topic at present, but current studies often focus on the host-guest interaction between CDs and hydrophobic substances, while ignoring the self-assembly behavior of the host and guest during the inclusion process. Based on the spatial structures, six representative terpenes were selected in this study, and the stoichiometric ratio between terpenes and β-cyclodextrin (β-CD) was investigated by isothermal titration calorimetry (ITC) method. Meanwhile, the constructed inclusion complexes (ICs) were characterized by nuclear magnetic resonance, X-ray diffraction, scanning electron microscope, etc. On this basis, the assembly process of β-CD and terpenes was investigated firstly by molecular simulation method, and the inclusion process between β-CD and terpenes was further studied in terms of the formation of individual ICs and ICs aggregates. Results showed that hydrophobic interaction and van der Waals force were the main driving forces of the reaction between terpenes and β-CD, and the formation of ICs, β-CD molecule aggregation, terpene aggregation, and ICs aggregation occurred simultaneously during the inclusion process of terpenes and β-CD. To sum up, this study may provide certain thinking and reference for the subsequent supramolecular chemical research on CDs.

PHD-BAH Domain in ASH1L Could Recognize H3K4 Methylation and Regulate the Malignant Behavior of Cholangio Carcinoma.

Histone methyltransferase absent, small, or homeotic discs1-like (ASH1L) is composed of su(var)3-9, enhancer of zeste, trithorax (SET) domain, pleckstrin homology domain (PHD) domain, middle (MID) domain, and bromo adjacent homology (BAH) domain. The SET domain of ASH1L is known to mediate mediate H3K36 dimethylation (H3K36me2) modification. However, the specific functions of the PHD-BAH domain remain largely unexplored. This study aimed to explore the biological function of the PHD-BAH domain in ASH1L. We employed a range of techniques, including a prokaryotic fusion protein expression purification system, pull-down assay, Isothermal Titration Calorimetry (ITC), polymerase chain reaction (PCR), and sitedirected mutagenesis, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR-Cas9) gene editing, cell culture experiment, western blot, cell proliferation assay, and cell apoptosis test. The PHD-BAH domain in ASH1L preferentially binds to the H3K4me2 peptide over H3K4 monomethylation (H3K4me1) and H3K4 trimethylation (H3K4me3) peptide. Notably, the W2603A mutation within the PHD-BAH domain could disrupt the interaction with H3K4me2 in vitro. Compared with wild-type Cholangiocarcinoma (CHOL) cells, deletion of the PHD-BAH domain in ASH1L led to increased CHOL cell apoptosis and reduced cell proliferation (P < 0.001). Additionally, the W2603A mutation affected the regulation of the proteasome 20S subunit beta (PSMB) family gene set. W2603A mutation was crucial for the interaction between the PHD-BAH domain and the H3K4me2 peptide. ASH1L regulated CHOL cell survival and proliferation through its PHD-BAH domain by modulating the expression of the PSMB family gene set.

A synthetic antibiotic class with a deeply-optimized design for overcoming bacterial resistance

The lack of new drugs that are effective against antibiotic-resistant bacteria has caused increasing concern in global public health. Based on this study, we report development of a modified antimicrobial drug through structure-based drug design (SBDD) and modular synthesis. The optimal modified compound, F8, was identified, which demonstrated in vitro and in vivo broad-spectrum antibacterial activity against drug-resistant bacteria and effectively mitigated the development of resistance. F8 exhibits significant bactericidal activity against bacteria resistant to antibiotics such as methicillin, polymyxin B, florfenicol (FLO), doxycycline, ampicillin and sulfamethoxazole. In a mouse model of drug-resistant bacteremia, F8 was found to increase survival and significantly reduce bacterial load in infected mice. Multi-omics analysis (transcriptomics, proteomics, and metabolomics) have indicated that ornithine carbamoyl transferase (arcB) is a antimicrobial target of F8. Further molecular docking, Isothermal Titration Calorimetry (ITC), and Differential Scanning Fluorimetry (DSF) studies verified arcB as a effective target for F8. Finally, mechanistic studies suggest that F8 competitively binds to arcB, disrupting the bacterial cell membrane and inducing a certain degree of oxidative damage. Here, we report F8 as a promising candidate drug for the development of antibiotic formulations to combat antibiotic-resistant bacteria-associated infections.

Structure-function analysis of nucleotide housekeeping protein HAM1 from human malaria parasite Plasmodium falciparum.

Non-canonical nucleotides, generated as oxidative metabolic by-products, significantly threaten the genome integrity of Plasmodium falciparum and thereby, their survival, owing to their mutagenic effects. PfHAM1, an evolutionarily conserved inosine/xanthosine triphosphate pyrophosphohydrolase, maintains nucleotide homeostasis in the malaria parasite by removing non-canonical nucleotides, although structure-function intricacies are hitherto poorly reported. Here, we report the X-ray crystal structure of PfHAM1, which revealed a homodimeric structure, additionally validated by size-exclusion chromatography-multi-angle light scattering analysis. The two monomeric units in the dimer were aligned in a parallel fashion, and critical residues associated with substrate and metal binding were identified, wherein a notable structural difference was observed in the β-sheet main frame compared to human inosine triphosphate pyrophosphatase. PfHAM1 exhibited Mg++-dependent pyrophosphohydrolase activity and the highest binding affinity to dITP compared to other non-canonical nucleotides as measured by isothermal titration calorimetry. Modifying the pfham1 genomic locus followed by live-cell imaging of expressed mNeonGreen-tagged PfHAM1 demonstrated its ubiquitous presence in the cytoplasm across erythrocytic stages with greater expression in trophozoites and schizonts. Interestingly, CRISPR-Cas9/DiCre recombinase-guided pfham1-null P. falciparum survived in culture under standard growth conditions, indicating its assistive role in non-canonical nucleotide clearance during intra-erythrocytic stages. This is the first comprehensive structural and functional report of PfHAM1, an atypical nucleotide-cleansing enzyme in P. falciparum.

Multianalytical Approach to Understand Polyphenol-Mal d 1 Interactions to Predict Their Impact on the Allergenic Potential of Apples.

Interactions between phenolic compounds and the allergen Mal d 1 are discussed to be the reason for better tolerance of apple cultivars, which are rich in polyphenols. Because Mal d 1 is susceptible to proteolytic digestion and allergenic symptoms are usually restricted to the mouth and throat area, the release of native Mal d 1 during the oral phase is of particular interest. Therefore, we studied the release of Mal d 1 under different in vitro oral digestion conditions and revealed that only 6-15% of the total Mal d 1 present in apples is released. To investigate proposed polyphenol-Mal d 1 interactions, various analytical methods, e.g., isothermal titration calorimetry, 1H-15N-HSQC NMR, and untargeted mass spectrometry, were applied. For monomeric polyphenols, only limited noncovalent interactions were observed, whereas oligomeric polyphenols and browning products caused aggregation. While covalent modifications were not detectable in apple samples, a Michael addition of epicatechin at cysteine 107 in r-Mal d 1.01 was observed.

Expression, Purification and Biophysical Characterisation of Klebsiella Pneumoniae Protein Adenylyltransferase: A Systematic Integration of Empirical and Computational Modelling Approaches.

Infections that are acquired due to a prolonged hospital stay and manifest 2 days following the admission of a patient to a health-care institution can be classified as hospital-acquired infections. Klebsiella pneumoniae (K. pneumoniae) has become a critical pathogen, posing serious concern globally due to the rising incidences of hypervirulent and carbapenem-resistant strains. Glutaredoxin is a redox protein that protects cells from oxidative stress as it associates with glutathione to reduce mixed disulfides. Protein adenylyltransferase (PrAT) is a pseudokinase with a proposed mechanism of transferring an AMP group from ATP to glutaredoxin. Inducing oxidative stress to the bacterium by inhibiting the activity of PrAT is a promising approach to combating its contribution to hospital-acquired infections. Thus, this study aims to overexpress, purify, and analyse the effects of ATP and Mg2+ binding to Klebsiella pneumoniae PrAT (KpPrAT). The pET expression system and nickel affinity chromatography were effective in expressing and purifying KpPrAT. Far-UV CD spectroscopy demonstrates that the protein is predominantly α-helical, even in the presence of Mg2+. Extrinsic fluorescence spectroscopy with ANS indicates the presence of a hydrophobic pocket in the presence of ATP and Mg2+, while mant-ATP studies allude to the potential nucleotide binding ability of KpPrAT. The presence of Mg2+ increases the thermostability of the protein. Isothermal titration calorimetry provides insight into the binding affinity and thermodynamic parameters associated with the binding of ATP to KpPrAT, with or without Mg2+. Conclusively, the presence of Mg2+ induces a conformation in KpPrAT that favours nucleotide binding.

Comparative Analysis Highlights Uniconazole's Efficacy in Enhancing the Cold Stress Tolerance of Mung Beans by Targeting Photosynthetic Pathways.

Soybean (Glycine max) and mung bean (Vigna radiata) are key legumes with global importance, but their mechanisms for coping with cold stress-a major challenge in agriculture-have not been thoroughly investigated, especially in a comparative study. This research aimed to fill this gap by examining how these two major legumes respond differently to cold stress and exploring the role of uniconazole, a potential stress mitigator. Our comprehensive approach involved transcriptomic and metabolomic analyses, revealing distinct responses between soybean and mung bean under cold stress conditions. Notably, uniconazole was found to significantly enhance cold tolerance in mung bean by upregulating genes associated with photosynthesis, while its impact on soybean was either negligible or adverse. To further understand the molecular interactions, we utilized advanced machine learning algorithms for protein structure prediction, focusing on photosynthetic pathways. This enabled us to identify LOC106780309 as a direct binding target for uniconazole, confirmed through isothermal titration calorimetry. This research establishes a new comparative approach to explore how soybean and mung bean adapt to cold stress, offers key insights to improve the hardiness of legumes against environmental challenges, and contributes to sustainable agricultural practices and food security.

Alkanols Regulate the Fluidity of Phospholipid Bilayer in Accordance to Their Concentration and Polarity.

In spite of the widespread use of alkanols as penetration enhancers, their effect on vesicular formulations remains largely unexplored. These can affect the stability and integrity of the phospholipid bilayers. In this study, we have investigated the interaction of linear (ethanol, butanol, hexanol, octanol) and branched alkanols (t-amylol and t-butanol) with three phospholipids (soya lecithin, SL; soy L-α-phosphatidylcholine, SPC; and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, DPPC). Thermodynamic and structural aspects of these interactions were studied as a function of the alkanol concentration and chain length. Our interpretations are based on isothermal titration calorimetry (ITC) and dynamic light scattering (DLS) experiments. We observed one-site interactions wherein hydroxyl and acyl groups interacted with the polar and nonpolar regions of the phospholipid, respectively. The stability and structural integrity of bilayers appeared to be dependent upon (a) the hydrocarbon chain length and concentration of alcohols, and (b) the degree of unsaturation in the phospholipid molecule. We found that these interactions triggered a reduction in the enthalpy which was compensated by increased entropy, keeping free energy negative. Drop in enthalpy indicates reversible disordering of the bilayer which enables the diffusion of alcohol without triggering destabilization. Ethanol engaged predominantly with the interface, and it resulted in higher enthalpic changes. Interactions became increasingly unfavorable with longer alcohols - a cutoff point was recorded with hexanol. The overall sequence of membrane disordering capability was recorded as follows: ethanol < butanol < octanol < hexanol. Octanol's larger size restricted its penetration in the bilayer, and hence it caused less enthalpic changes relative to hexanol. This could also be verified from the trends in the area ratio of these vesicles obtained from the DLS data. Branched alkanols displayed a lower binding affinity with the phospholipids relative to their linear counterparts. These data are useful while contemplating the inclusion of short-chain alcohols as penetration enhancers in phospholipid vesicles.

Comparative Analysis Highlights Uniconazole’s Efficacy in Enhancing the Cold Stress Tolerance of Mung Beans by Targeting Photosynthetic Pathways

Soybean (Glycine max) and mung bean (Vigna radiata) are key legumes with global importance, but their mechanisms for coping with cold stress—a major challenge in agriculture—have not been thoroughly investigated, especially in a comparative study. This research aimed to fill this gap by examining how these two major legumes respond differently to cold stress and exploring the role of uniconazole, a potential stress mitigator. Our comprehensive approach involved transcriptomic and metabolomic analyses, revealing distinct responses between soybean and mung bean under cold stress conditions. Notably, uniconazole was found to significantly enhance cold tolerance in mung bean by upregulating genes associated with photosynthesis, while its impact on soybean was either negligible or adverse. To further understand the molecular interactions, we utilized advanced machine learning algorithms for protein structure prediction, focusing on photosynthetic pathways. This enabled us to identify LOC106780309 as a direct binding target for uniconazole, confirmed through isothermal titration calorimetry. This research establishes a new comparative approach to explore how soybean and mung bean adapt to cold stress, offers key insights to improve the hardiness of legumes against environmental challenges, and contributes to sustainable agricultural practices and food security.

Peptide-based CE-SELEX enables convenient isolation of aptamers specifically recognizing CD20-expressing cells

The CD20 antigen is a key target for several diseases including lymphoma and autoimmune diseases. For over 20 years, several monoclonal antibodies were developed to treat CD20-related disorders. As many therapeutic proteins, their clinical use is however limited due to their nature with a costly biotechnological procedure and side effects such as the production of anti-drug neutralizing antibodies. Nucleic acid aptamers have some advantages over mAbs and are currently investigated for clinical use. We herein report the selection of DNA aptamer by using a peptide-based CE-SELEX (Capillary Electrophoresis-Systematic Evolution of Ligands by Exponential Enrichment) method. It was demonstrated that these aptamers bind specifically a CD20-expressing human cell line, with Kd estimated from isothermal titration calorimetry experiments in the micromolar range. This study demonstrates that the CE-SELEX is suitable as alternative method to the conventional Cell-SELEX to discover new cell-targeting compounds.

Calibrating ITC instruments: Problems with weak base neutralization

Modern isothermal titration calorimetry instruments give great precision, but for comparable accuracy they require chemical calibration. For the heat factor, one recommended process is HCl into the weak base TRIS. In studying this reaction with a VP-ITC and two Nano-ITCs, we have encountered some problems, most importantly a titrant volume shortfall Δv ≈ 0.3 μL, which we attribute to diffusive loss of HCl in the syringe tip. This interpretation is supported by a mathematical treatment of the diffusion problem. The effect was discovered through a variable-v protocol, which thus should be used to properly allow for it in any reaction that similarly approaches completion. We also find that the effects from carbonate contamination and from OH− from weak base hydrolysis can be more significant that previously thought. To facilitate proper weighting in the least-squares fitting of data, we have estimated data variance functions from replicate data. All three instruments have low-signal precision of σ ≈ 1 μJ; titrant volume uncertainty is a factor of ∼2 larger for the Nano-ITCs than for the VP-ITC. The final heat factors remain uncertain by more than the ∼1 % precision of the instruments and are unduly sensitive to the HCl concentration.

Development of an effective meropenem/KPC-2 inhibitor combination to combat infections caused by carbapenem-resistant K. pneumoniae

The global public health threat of bacterial antibiotic resistance continues to escalate and necessitates the implementation of urgent measures to expand our arsenal of antimicrobial drugs. In this study, we identified a benzoxaborane compound, namely 5-chloro-1,3-dihydro-1-hydroxy-2,1-benzoxaborole (AN2178), which can effectively inhibit the catalytic activity of the Klebsiella pneumoniae carbapenemase (KPC-2) enzyme. The efficacy of AN2718 as an inhibitor for the KPC-2 enzyme was verified through various assays, including enzyme activity assays and isothermal titration calorimetry. Results of multiple biochemical assays, minimum inhibitory concentration assay, and time-killing assay also showed that binding of AN2718 to KPC-2 enabled the restoration of the bactericidal effect of meropenem. The survival rate of mice infected by carbapenem-resistant, high-virulence strains increased significantly upon treatment with this agent. Most importantly, the meropenem and AN2718 combination is effective on KPC-2 mutations such as KPC-33 that were clinically evolved and exhibited resistance to ceftazidime-avibactam upon the clinical uses of this drug for a couple of years. Comprehensive safety tests both in vitro and in vivo, such as cytotoxicity, haemolytic activity, and cytochrome P450 inhibition assays demonstrated that AN2718 was safe for clinical use. These promising data indicate that AN2718 has a high potential for being approved for the treatment of drug resistant bacterial infections, including those caused by the Ceftazidime-Avibactam resistant strains. To conclude, the compound AN2718 can be regarded as a valuable addition to the current antimicrobial armamentarium and a promising tool to combat antimicrobial resistance.