Bengt Erik Haug

Bacterial biofilms pose a major public health challenge by increasing the antimicrobial tolerance in pathogenic bacteria, thereby limiting the effect of medication-based treatment and promoting the development of antimicrobial resistance. Hence, there is a need to discover new molecules with the ability to prevent biofilm formation. We screened seven synthetic analogues of the breitfussin family of natural products for antimicrobial and antibiofilm activity using a broth microdilution and crystal violet method, respectively. Two compounds inhibited the growth of Gram-positive bacteria in their planktonic state at concentrations of 50 µM, of which one compound (2) demonstrated the ability to inhibit the biofilm formation of Staphylococcus epidermidis at sub-growth-inhibitory, low micromolar concentrations. Compound 2 did not inhibit biofilm growth in Staphylococcus aureus or Listeria monocytogenes, or the ability to eradicate pre-established biofilms. Initial Mode of Action (MoA) studies with compound 2 against S. epidermidis showed a modest impact on the cell surface hydrophobicity and early-stage adhesion to polystyrene. These findings highlight the breitfussin framework as a promising scaffold for the development of new antimicrobial and antibiofilm agents.

Voltage-gated potassium channel KCNQ1 (Kv7.1) plays a critical role in electrical excitability in the heart, gut, and brain. Together with the auxiliary subunit KCNE1, KCNQ1 generates a slow delayed rectifier current (I_Ks) that is essential for cardiac repolarization. Mutations and dysregulation of this channel are found in channelopathies leading to sudden death, long-QT syndrome, atrial fibrillation, epilepsy, deafness, diabetes, and neuropsychiatric disorders. Although KCNQ1 and related potassium channels are promising therapeutic targets, there are few potent, selective, and therapeutically safe inhibitors and activators available for these proteins. A virtual screening of 36,374 compounds was conducted against KCNQ1, followed by in silico analyses that identified eight potential ligand candidates for experimental evaluation using human KCNQ1 coexpressed with KCNE1 in Xenopus laevis oocytes. Electrophysiological recordings showed that the benzodiazepine-based ligand Zinc13732787 was a potent inhibitor of the channel complex, without affecting KCNQ2/KCNQ3. Based on virtual screening and molecular docking, the 1-(3-chlorophenyl)-urea substituent on the benzodiazepine core is important for selective inhibition of KCNQ1/KCNE1, as further supported by structure-activity relationship and stereochemical exploration of Zinc13732787. Furthermore, low concentrations of Zinc13732787 reduced neurite outgrowth in human neuronal stem cells (NSCs), mirroring the phenotype observed in homozygous KCNQ1-knockout cells. Importantly, Zinc13732787 did not affect NSC proliferation, nor did it induce cytotoxicity. In homozygous KCNQ1-knockout NSCs, compound Zinc13732787 had no effect on neurite outgrowth, indicating high target specificity. These findings suggest that this compound is a valuable tool for investigating the physiological and pathological roles of KCNQ1 across various tissues. Additionally, it could be used as a precursor for novel antiarrhythmic agents as well as for epilepsy and neuropsychiatric conditions.

A metal-free, one-pot synthesis of 2,3-unsubstituted indoles via an adapted Leimgruber–Batcho protocol has been developed.

Sewage sludge contains active pharmaceutical ingredients (APIs) that persist after anaerobic digestion, posing challenges for its direct use as an organic amendment. This study presents a proof-of-concept investigation of the degradation of APIs through hydrothermal liquefaction (HTL), supporting the sustainability of using digested sewage sludge (DSS) as a feedstock for bio-oil production. In this work, the degradation of the four APIs carbamazepine, sertraline, telmisartan, and citalopram during HTL was investigated. Two experimental series were conducted at 330 °C for 4 h: one with APIs and formic acid only, and one with APIs, formic acid, and DSS. Formic acid was added to the reaction system as a hydrogen donor. Quantitative analyses of the organic product phases using GC–MS revealed complete degradation of all four APIs. The proposed degradation schemes provide detailed insights into the degradation pathways of the APIs during HTL, and the results suggest a greatly reduced likelihood of remaining pharmacological activity in the resulting products. These findings represent a significant step towards understanding and mitigating the risks associated with APIs in DSS and encourage further research to optimise the HTL process.

A combinatorial approach integrating BLI and ligand-based NMR for RNA-focused fragment-based drug discovery.

Neutrophil proteinase 3 (PR3) is an important drug target for inflammatory lung diseases such as chronic obstructive pulmonary disease and cystic fibrosis. Drug discovery efforts targeting PR3 require active enzyme for in vitro characterization, such as inhibitor screening, enzymatic assays, and structural studies. Recombinant expression of active PR3 overcomes the need for enzyme supplies from human blood and in addition allows studies on the influence of mutations on enzyme activity and ligand binding. Here, we report the expression of recombinant PR3 (rPR3) using a baculovirus expression system. The purification and activation process described resulted in highly pure and active PR3. The activity of rPR3 in the presence of commercially available inhibitors was compared with human PR3 by using a fluorescence-based enzymatic assay. Purified rPR3 had comparable activity to the native human enzyme, thus being a suitable alternative for enzymatic studies in vitro. Further, we established a surface plasmon resonance-based assay to determine binding affinities and kinetics of PR3 ligands. These methods provide valuable tools for early drug discovery aiming towards treatment of lung inflammation.

The role of aromatic amino acids in peripheral protein membrane binding has been reported to involve cation–π interactions with choline lipids. In this study, we have investigated the interactions of the model pentapeptide Ac-WL-X-LL-OH (where X = L, Y, F, or W) with the phospholipid membrane using solid-state NMR. The effect of guest residue X on the peptide-lipid interactome was complementary to the seminal report on the interfacial hydrophobicity scale by Wimley and White. We found that the phospholipids retained a lamellar phase in the presence of each of the peptides with an aromatic X residue, whereas the Leu peptide perturbed the bilayer to an extent where an additional isotropic phase was observed. The solid-state NMR ¹³C and ³¹P data provide additional information on the influence of these short peptides on the membrane that has not been previously reported. The magnitude of membrane perturbation was in the order of guest residue X = L > Y~F > W, which is consistent with the relative amino acid interfacial affinity reported by Wimley and White. Further work is, however, required to uncover the behavior of the peptide and localization in the membrane domain due to ambiguity of the ¹³C NMR data. We have launched efforts in this regard for the objective of better understanding the role of aromatic amino acids in peripheral membrane protein binding.

Using a combination of multisite lambda-dynamics (MS lambda D) together with in vitro IC50 assays, we evaluated the polypharmacological potential of a scaffold currently in clinical trials for inhibition of human neutrophil elastase (HNE), targeting cardiopulmonary disease, for efficacious inhibition of Proteinase 3 (PR3), a related neutrophil serine proteinase. The affinities we observe suggest that the dihydropyrimidinone scaffold can serve as a suitable starting point for the establishment of polypharmacologically targeting both enzymes and enhancing the potential for treatments addressing diseases like chronic obstructive pulmonary disease.

There is an urgent need for new antibiotics. FabF (3-oxoacyl-[acyl-carrier-protein] synthase 2), which catalyses the rate limiting condensation reaction in the fatty acid synthesis II pathway, is an attractive target. Very few inhibitors of FabF are known and most are derived from natural products. In an effort to further explore the chemical space of FabF ligands, we have carried out fragment screening by X-ray crystallography against an intermediated state-mimicking variant of P. aeruginosa FabF (PaFabF C164Q). This screen has screen resulted in 16 hits binding in or close to the malonyl-CoA or fatty acid binding site or an adjacent dimer interface. More than 80 analogues of the hit compounds have been explored making use of either commercially available or synthesised compounds. For 8 of those co-crystal structures could be determined and the most potent ligand has a binding affinity of 65 µM. This data package forms a strong foundation for the development of more potent and diverse FabF inhibitors.

Using a combination of state-of-the-art free energy computational approaches together with in vitro IC50 assays, we evaluated the polypharmacological potential of a scaffold currently in clinical trials for inhibition of human neutrophil elastase (HNE), targeting cardiopulmonary disease, for efficacious inhibition of Proteinase 3 (PR3), a related neutrophil serine proteinase. Based on benchmarking of a series of ligands with known inhibitory activity against HNE using multisite λ-dynamics (MSλD) and multiple force field models, we demonstrated the best agreement between measured IC50 values and computed binding free energies using a hybrid force field of CHARMM c36m for the protein and ligand parameters from OPLS LigParGen that utilizes a CM1A-LBCC charge model. Using this computational model, the ligands were assessed as potential inhibitors of PR3 by anchoring the computed relative binding affinities to a single measured IC50. This led to suggested potential additional inhibitors for PR3 from those tested and characterized to be good binders to HNE. We synthesized and assayed a number of these compounds to further test our predicted binding affinities and discovered that both prediction and experiment demonstrated that the compounds bound less well to PR3, we observed a range of IC50s from 7.7-190 nM in PR3 to 0.5-1.3 nM for the same inhibitors for HNE. However, the affinities we observe suggest that the scaffold can serve as a suitable starting point for the establishment of polypharmacologically targeting both enzymes and enhancing the potential for treatments addressing diseases like chronic obstructive pulmonary disease.