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Tick-borne encephalitis (TBE) is a severe neurological disorder caused by tick-borne encephalitis virus (TBEV), a member of the Flavivirus genus. Currently, two vaccines are available in Europe against TBEV. However, TBE cases have been rising in Sweden for the past twenty years, and thousands of cases are reported in Europe, emphasizing the need for antiviral treatments against this virus. The NS2B-NS3 protease is essential for flaviviral life cycle and has been studied as a target for the design of inhibitors against several well-known flaviviruses, but not TBEV. In the present study, Compound 86, a known tripeptidic inhibitor of dengue (DENV), West Nile (WNV) and Zika (ZIKV) proteases, was predicted to be active against TBEV protease using a combination of in silico techniques. Further, Compound 86 was found to inhibit recombinant TBEV protease with an IC50 = 0.92 mu M in the in vitro enzymatic assay. Additionally, two more peptidic analogues were synthetized and they displayed inhibitory activities against both TBEV and ZIKV proteases. In particular, Compound 104 inhibited ZIKV protease with an IC50 = 0.25 mu M. These compounds represent the first reported inhibitors of TBEV protease to date and provides valuable information for the further development of TBEV as well as pan-flavivirus protease inhibitors.

Zika virus (ZIKV) is an emerging mosquito-borne flavivirus and infection by ZIKV Asian lineage is known to cause fetal brain anomalies and Guillain-Barrés syndrome. The WHO declared ZIKV a global public health emergency in 2016. However, currently neither vaccines nor antiviral prophylaxis/treatments are available. In this study, we report the identification of a C2-symmetric diol-based Human immunodeficiency virus type-1 (HIV) protease inhibitor active against ZIKV NS2B-NS3 protease. The compound, referred to as 9b, was identified by in silico screening of a library of 6265 protease inhibitors. Molecular dynamics (MD) simulation studies revealed that compound 9b formed a stable complex with ZIKV protease. Interaction analysis of compound 9b's binding pose from the cluster analysis of MD simulations trajectories predicted that 9b mostly interacted with ZIKV NS3. Although designed as an aspartyl protease inhibitor, compound 9b was found to inhibit ZIKV serine protease in vitro with IC₅₀ = 143.25 ± 5.45 µM, in line with the in silico results. Additionally, linear interaction energy method (LIE) was used to estimate binding affinities of compounds 9b and 86 (a known panflavivirus peptide hybrid with IC₅₀ = 1.64 ± 0.015 µM against ZIKV protease). The LIE method correctly predicted the binding affinity of compound 86 to be lower than that of 9b, proving to be superior to the molecular docking methods in scoring and ranking compounds. Since most of the reported ZIKV protease inhibitors are positively charged peptide-hybrids, with our without electrophilic warheads, compound 9b represents a less polar and more drug-like non-peptide hit compound useful for further optimization.Communicated by Ramaswamy Sarma.

In vitro screening of large compound libraries with automated high-throughput screening is expensive and time-consuming and requires dedicated infrastructures. Conversely, the selection of DNA-encoded chemical libraries (DECLs) can be rapidly performed with routine equipment available in most laboratories. In this study, we identified novel inhibitors of SARS-CoV-2 main protease (M^pro) through the affinity-based selection of the DELopen library (open access for academics), containing 4.2 billion compounds. The identified inhibitors were peptide-like compounds containing an N-terminal electrophilic group able to form a covalent bond with the nucleophilic Cys145 of M^pro, as confirmed by x-ray crystallography. This DECL selection campaign enabled the discovery of the unoptimized compound SLL11 (IC₅₀ = 30 nM), proving that the rapid exploration of large chemical spaces enabled by DECL technology allows for the direct identification of potent inhibitors avoiding several rounds of iterative medicinal chemistry. As demonstrated further by x-ray crystallography, SLL11 was found to adopt a highly unique U-shaped binding conformation, which allows the N-terminal electrophilic group to loop back to the S1′ subsite while the C-terminal amino acid sits in the S1 subsite. MP1, a close analog of SLL11, showed antiviral activity against SARS-CoV-2 in the low micromolar range when tested in Caco-2 and Calu-3 (EC₅₀ = 2.3 µM) cell lines. As peptide-like compounds can suffer from low cell permeability and metabolic stability, the cyclization of the compounds will be explored in the future to improve their antiviral activity.

Herein, we describe a convenient protocol for the synthesis of N-alkenylated heterocycles using abundant ketone electrophiles and T3P as a water scavenger under microwave irradiation. The method can be applied to a diverse range of NH-heterocycles and ketones with good to excellent yields (up to 94%). This procedure is particularly attractive, as it is metal- and base-free, tolerates a variety of functional groups, and offers ease of product purification. The utility of the protocol was exemplified by synthesizing pharmaceutically relevant scaffolds containing the N-alkenyl motif and was further extended to a one-pot reductive amination sequence.

Tricyclic antidepressants (TCA) are among the oldest antidepressants that are also indicated for the treatment of various pain conditions. This drug class includes cationic amphiphilic molecules characterized by a positively charged hydrophilic head group. Microgels consist of cross-linked polymers and can act as drug delivery systems that protect drugs from degradation, as well as release drugs when exposed to high ionic strength. Studying these drug-microgel interactions using microfluidics is beneficial as it reduces product drop and costs. Therefore, the aim of this study is to investigate with a microfluidics-based method how the microgels (Hyaluronic acid (HA), Polyacrylate (PA) and DC beads) with a scale of 100-300 mm in diameter interact with the amphiphilic antidepressant drugs (Amitriptyline (AMT), Doxepin (DOX) and Chlorpromazine (CPZ)), as well as investigating whether the aforementioned gels could function as drug formulations. The experiments were divided into two studies binding and release studies. Binding studies were done for different concentrations of AMT, DOX and CPZ. Binding studies of AMT, DOX and CPZ to PA gels, HA gels and DC beads only took place in phosphate buffer. For amphiphilic drugs, a Critical Association Concentration (CAC) and Critical collapse concentration (CCC) were first determined where the kinetics of binding and release were carefully studied. This work's results showed similarities and differences between AMT, CPZ, and DOX interacting with DC, PA, and HA gels by using microfluidics-based method. Lower concentration of substance caused less electrostatic interactions between drug and the microgels, leading to less shrinkage of microgels. The higher the net charge of a drug, the more electrostatic interactions with microgels, resulting in slower release. Therefore, microgel volume change is time-dependent for both binding and release. The ion-binding property of microgels is particularly useful in drug delivery, where linear and cross-linked former of the negatively charged polymer can act as carriers of cationic self-assembled drugs, it tells us that microgels can be used as drug formulations. 

Macrocyclic analogues of the linear hexapeptide, angiotensin IV (AngIV) have proved to be potent inhibitors of insulin-regulated aminopeptidase (IRAP, oxytocinase, EC 3.4.11.3). Along with higher affinity, macrocycles may also offer better metabolic stability, membrane permeability and selectivity, however predicting the outcome of particular cycle modifications is challenging. Here we describe the development of a series of macrocyclic IRAP inhibitors with either disulphide, olefin metathesis or lactam bridges and variations of ring size and other functionality. The binding mode of these compounds is proposed based on molecular dynamics analysis. Estimation of binding affinities (∆G) and relative binding free energies (∆∆G) with the linear interaction energy (LIE) method and free energy perturbation (FEP) method showed good general agreement with the observed inhibitory potency. Experimental and calculated data highlight the cumulative importance of an intact N-terminal peptide, the specific nature of the macrocycle, the phenolic oxygen and the C-terminal functionality.

Herein, we present the design and synthesis of 2(1H)-pyrazinone based HCV NS3 protease inhibitors and show that elongated R-3 urea substituents were associated with increased inhibitory potencies over several NS3 protein variants. The inhibitors are believed to rely on beta-sheet mimicking hydrogen bonds which are similar over different genotypes and current drug resistant variants and correspond to the beta-sheet interactions of the natural peptide substrate. Inhibitor 36, for example, with a urea substituent including a cyclic imide showed balanced nanomolar inhibitory potencies against genotype la, both wild-type (K-i=30 nM) and R155K (K-i=2 nM), and genotype 3a (K-i=5 nM).

Oligopeptide boronates with a lipophilic tail are known to inhibit the type I signal peptidase in E. coli, which is a promising drug target for developing novel antibiotics. Antibacterial activity depends on these oligopeptides having a cationic modification to increase their permeation. Unfortunately, this modification is associated with cytotoxicity, motivating the need for novel approaches. The sulfonimidamide functionality has recently gained much interest in drug design and discovery, as a means of introducing chirality and an imine-handle, thus allowing for the incorporation of additional substituents. This in turn can tune the chemical and biological properties, which are here explored. We show that introducing the sulfonimidamide between the lipophilic tail and the peptide in a series of signal peptidase inhibitors resulted in antibacterial activity, while the sulfonamide isostere and previously known non-cationic analogs were inactive. Additionally, we show that replacing the sulfonamide with a sulfonimidamide resulted in decreased cytotoxicity, and similar results were seen by adding a cationic sidechain to the sulfonimidamide motif. This is the first report of incorporation of the sulfonimidamide functional group into bioactive peptides, more specifically into antibacterial oligopeptides, and evaluation of its biological effects.

New antibacterial compounds are urgently needed, especially for infections caused by the top-priority Gram-negative bacteria that are increasingly difficult to treat. Lipid A is a key component of the Gram-negative outer membrane and the LpxH enzyme plays an important role in its biosynthesis, making it a promising antibacterial target. Inspired by previously reported ortho-N-methyl-sulfonamidobenzamide-based LpxH inhibitors, novel benzamide substitutions were explored in this work to assess their in vitro activity. Our findings reveal that maintaining wild-type antibacterial activity necessitates removal of the N-methyl group when shifting the ortho-N-methyl-sulfonamide to the meta-position. This discovery led to the synthesis of meta-sulfonamidobenzamide analogs with potent antibacterial activity and enzyme inhibition. Moreover, we demonstrate that modifying the benzamide scaffold can alter blocking of the cardiac voltage-gated potassium ion channel hERG. Furthermore, two LpxH-bound X-ray structures show how the enzyme-ligand interactions of the meta-sulfonamidobenzamide analogs differ from those of the previously reported ortho analogs. Overall, our study has identified meta-sulfonamidobenzamide derivatives as promising LpxH inhibitors with the potential for optimization in future antibacterial hit-to-lead programs.

With the objective of finding new classes of cognitive enhancers with potential for the treatment of neurodegenerative disorders, such as Alzheimer's disease, small molecule inhibitors of insulin-regulated aminopeptidase (IRAP) were designed and synthesized. IRAP is a member of the M1 family of zinc aminopeptidases and is abundantly expressed in areas of the brain associated with cognition, such as the amygdala, hippocampus and cerebral cortex. IRAP inhibitors were previously shown to enhance memory and learning in animal models. A comprehensive high throughput screening of 400,000 small molecules from the European Lead Factory library provided a series of 50 promising compounds in a qualified hit list (QHL). More than 30 IRAP inhibitors with an IC₅₀ below 3.5 μM were identified. Herein, selected compounds from this QHL were assayed for solubility and permeability. Most of the selected compounds displayed good solubility, but further permeability studies on the best compounds revealed low blood brain barrier (BBB) permeability and high efflux in cells overexpressing P-gp pumps, rendering them less promising as starting points in drug discovery processes. Two compounds from the QHL were prioritized for further structural optimization; the pyridyl-substituted isoxazole 1a (QHL27) and the benzylhydroxamic acid derivative 1b (QHL1), both demonstrating fair BBB permeability and no indication of efflux. While our attempts to improve the isoxazole derivative 1a were not fruitful, a structural modification of 1b to provide the chloro-substituted benzylhydroxamic acid 14b resulted in a ten-fold improvement of the IRAP inhibition with an IC₅₀ value of 60 nM. The binding modes of 1b and 14b were determined by free energy perturbation (FEP) analysis performed on candidate docking poses, determining a binding mode that accurately explained the experimental SAR. Further FEP studies of compound 14b suggested that it exhibits selectivity towards IRAP over Aminopeptidase N (APN), indicating its potential for targeted therapeutic applications.

Introduction: The drug development cost is increasing and developing a successful drug is estimated to be in the range of billions of dollars. The leading causes for this are due to a general increase in R&D cost as well as a decrease in clinical success rates. In silico methods, such as virtual screenings, have become more prominent within drug discovery as they can early in development identify potential chemical starting points. However, with the massive increase in available compounds and molecular targets in the last decades, these datasets need to be properly filtered to generate good results. Recently, a tool called macHine leArning booSTEd dockiNg (HASTEN) has been implemented in virtual screening campaigns to enhance their effectiveness. By only docking a fraction of dataset, HASTEN can accurately predict docking scores for the remaining compounds, speeding up the screening process. Although HASTEN in its current state can generate good results, the tool is still being improved.

Aim: The aim of this study is to find optimizations in the HASTEN protocol to better predict top scoring compounds by investigating HASTEN’s performance on three antibiotic targets.

Methods: Baseline performance was generated by training models using the default HASTEN settings, where recall was used as the primary evaluation metric. New models were trained by systematically changing settings to improve HASTEN’s performance. Various settings were investigated, including the number of iterations, different datasets variants, training set size, additional feature generators, hyperparameter optimization, activation methods and optimizers.

Results: Many investigated settings failed to improve performance, while some even decreased performance. The biggest increase in model performance was seen in models trained with additional Morgan fingerprints, which improved recall by 20% across all targets. The addition of Morgan fingerprints made the models less stable but could be stabilized with the addition of hyperparameters.

Conclusions: In this study we found optimizations that could enhance the performance of HASTEN screenings as well as settings to avoid. Mainly, the addition of Morgan fingerprint features improved recall values the most, in conjunction with hyperparameters it created a stable and reliable model. Potential areas that could further improve HASTEN performance include investigation of larger datasets, systematic selection of initial training compounds, cross-fold validation and ensembles.

Carbapenems are vital antibiotics, but their efficacy is increasingly compromised by metallo-β-lactamases (MBLs). Here we report the discovery and optimization of potent broad-spectrum MBL inhibitors. A high-throughput screen for NDM-1 inhibitors identified indole-2-carboxylates (InCs) as potential β-lactamase stable β-lactam mimics. Subsequent structure-activity relationship studies revealed InCs as a new class of potent MBL inhibitor, active against all MBL classes of major clinical relevance. Crystallographic studies revealed a binding mode of the InCs to MBLs that, in some regards, mimics that predicted for intact carbapenems, including with respect to maintenance of the Zn(II)-bound hydroxyl, and in other regards mimics binding observed in MBL-carbapenem product complexes. InCs restore carbapenem activity against multiple drug-resistant Gram-negative bacteria and have a low frequency of resistance. InCs also have a good in vivo safety profile, and when combined with meropenem show a strong in vivo efficacy in peritonitis and thigh mouse infection models.

Here we describe the development of biaryl 2,5-diphenylphospholanes as a new class of C-2-symmetric, monodentate ligands for asymmetric Suzuki-Miyaura (ASM) reactions. Screening of a series of exemplary phospholanes led to the identification of two ligands that were used to prepare a range of atropisomeric biaryl and heterobiaryl products with good to excellent levels of enantioselectivity (up to 97:3 e.r.) under mild conditions. DFT studies suggest that the formation of a constraining ligand pocket and coordination of one of the biaryl methoxy groups in the optimised ligands to the metal centre is crucial for restricting conformational freedom in the bond-forming step.

The borylation of aryl and heteroaryl C–H bonds is valuable for the site-selective functionalization of C–H bonds in complex molecules. Iridium catalysts ligated by bipyridine ligands catalyze the borylation of the C–H bond that is most acidic and least sterically hindered in an arene, but predicting the site of borylation in molecules containing multiple arenes is difficult. To address this challenge, we report a hybrid computational model that predicts the Site of Borylation (SoBo) in complex molecules. The SoBo model combines density functional theory, semiempirical quantum mechanics, cheminformatics, linear regression, and machine learning to predict site selectivity and to extrapolate these predictions to new chemical space. Experimental validation of SoBo showed that the model predicts the major site of borylation of pharmaceutical intermediates with higher accuracy than prior machine-learning models or human experts, demonstrating that SoBo will be useful to guide experiments for the borylation of specific C(sp²)–H bonds during pharmaceutical development.

The tumor micro-environment (TME) of hepatocellular carcinoma (HCC) consists out of cirrhotic liver tissue and is characterized by an extensive deposition of extracellular matrix proteins (ECM). The evolution from a reversible fibrotic state to end-stage of liver disease, namely cirrhosis, is characterized by an increased deposition of ECM, as well as changes in the exact ECM composition, which both contribute to an increased liver stiffness and can alter tumor phenotype. The goal of this study was to assess how changes in matrix composition and stiffness influence tumor behavior. HCC-cell lines were grown in a biomimetic hydrogel model resembling the stiffness and composition of a fibrotic or cirrhotic liver. When HCC-cells were grown in a matrix resembling a cirrhotic liver, they increased proliferation and protein content, compared to those grown in a fibrotic environment. Tumour nodules spontaneously formed outside the gels, which appeared earlier in cirrhotic conditions and were significantly larger compared to those found outside fibrotic gels. These tumor nodules had an increased expression of markers related to epithelial-to-mesenchymal transition (EMT), when comparing cirrhotic to fibrotic gels. HCC-cells grown in cirrhotic gels were also more resistant to doxorubicin compared with those grown in fibrotic gels or in 2D. Therefore, altering ECM composition affects tumor behavior, for instance by increasing pro-metastatic potential, inducing EMT and reducing response to chemotherapy.

Chemotherapy causes intestinal mucositis, which includes villous atrophy and altered mucosal barrier function. However, there is an uncertainty regarding how the reduced small-intestinal surface area affects the mucosal permeability of the small marker probe mannitol (MW 188), and how the mucosa responds to luminal irritants after chemotherapy. The aims in this study were to determine (i) the relationship between chemotherapy-induced villus atrophy and the intestinal permeability of mannitol and (ii) how the mucosa regulate this permeability in response to luminal ethanol and sodium dodecyl sulfate (SDS). This was investigated by treating rats with a single intraperitoneal dose of doxorubicin, irinotecan, or 5-fluorouracil. After 72 h, jejunum was single-pass perfused and mannitol permeability determined at baseline and after 15 min luminal exposure to 15% ethanol or 5 mg/mL SDS. Tissue samples for morphological analyses were sampled from the perfused segment. All three chemotherapeutics caused a similar 30% reduction in villus length. Mannitol permeability increased with irinotecan (1.3-fold) and 5-fluorouracil (2.5-fold) and was reduced with doxorubicin (0.5-fold), suggesting that it is not epithelial surface area alone that regulates intestinal permeability to mannitol. There was no additional increase in mannitol permeability induced by luminal ethanol or SDS in the chemotherapy-treated rats compared to controls, which may be related to the relatively high basal permeability of mannitol compared to other common low-permeability probes. We therefore suggest that future studies should focus on elucidating the complex interplay between chemotherapy in combination with luminal irritants on the intestinal permeability of other probes.

The Sirtuin family of proteins has been found to play interesting roles in disease. Of this family, Sirtuin 2 (SIRT2) in particular has shown potential as a therapeutic target for different types of cancers and neurodegenerative diseases. Multiple inhibitors and probes have been developed to study it, in particular in the search for compounds with high selectivity to SIRT2. In recent years, the 1,2,4-oxadiazole class of compounds has been shown to produce highly active and selective compounds. In addition, a new simple method of synthesis was described in literature that allows for the simple synthesis of these compounds. This project aimed to utilize rigid docking to screen a large number of compounds to discover novel 1,2,4-oxadiazole-derived SIRT2 inhibitors that can be synthesized with the new protocol previously described. Promising binders were tested via MD simulations and compared using FEP calculations.

The Nrf2 transcription factor is a key regulator of redox reactions and considered the main target for the multiple sclerosis (MS) drug dimethyl fumarate (DMF). However, exploration of additional Nrf2-activating compounds is motivated, since DMF displays significant off-target effects and has a relatively poor penetrance to the central nervous system (CNS). We de novo synthesized eight vinyl sulfone and sulfoximine compounds (CH-1-CH-8) and evaluated their capacity to activate the transcription factors Nrf2, NF kappa B, and HIF1 in comparison with DMF using the pTRAF platform. The novel sulfoximine CH-3 was the most promising candidate and selected for further comparison in vivo and later an experimental model for traumatic brain injury (TBI). CH-3 and DMF displayed comparable capacity to activate Nrf2 and downstream transcripts in vitro, but with less off-target effects on HIF1 from CH-3. This was verified in cultured microglia and oligodendrocytes (OLs) and subsequently in vivo in rats. Following TBI, DMF lowered the number of leukocytes in blood and also decreased axonal degeneration. CH-3 preserved or increased the number of pre-myelinating OL. While both CH-3 and DMF activated Nrf2, CH-3 showed less off-target effects and displayed more selective OL associated effects. Further studies with Nrf2-acting compounds are promising candidates to explore potential myelin protective or regenerative effects in demyelinating disorders.