Rational design of balanced dual-targeting antibiotics with limited resistance.

Antibiotics that inhibit multiple bacterial targets offer a promising therapeutic strategy against resistance evolution, but developing such antibiotics is challenging. Here we demonstrate that a rational design of balanced multitargeting antibiotics is feasible by using a medicinal chemistry workflow. The resultant lead compounds, ULD1 and ULD2, belonging to a novel chemical class, almost equipotently inhibit bacterial DNA gyrase and topoisomerase IV complexes and interact with multiple evolutionary conserved amino acids in the ATP-binding pockets of their target proteins. ULD1 and ULD2 are excellently potent against a broad range of gram-positive bacteria. Notably, the efficacy of these compounds was tested against a broad panel of multidrug-resistant Staphylococcus aureus clinical strains. Antibiotics with clinical relevance against staphylococcal infections fail to inhibit a significant fraction of these isolates, whereas both ULD1 and ULD2 inhibit all of them (minimum inhibitory concentration [MIC] ≤1 µg/mL). Resistance mutations against these compounds are rare, have limited impact on compound susceptibility, and substantially reduce bacterial growth. Based on their efficacy and lack of toxicity demonstrated in murine infection models, these compounds could translate into new therapies against multidrug-resistant bacterial infections.

Structure-Function Studies of Sponge-Derived Compounds on the Cardiac CaV3.1 Channel.

T-type calcium (CaV3) channels are involved in cardiac automaticity, development, and excitation-contraction coupling in normal cardiac myocytes. Their functional role becomes more pronounced in the process of pathological cardiac hypertrophy and heart failure. Currently, no CaV3 channel inhibitors are used in clinical settings. To identify novel T-type calcium channel ligands, purpurealidin analogs were electrophysiologically investigated. These compounds are alkaloids produced as secondary metabolites by marine sponges, and they exhibit a broad range of biological activities. In this study, we identified the inhibitory effect of purpurealidin I (1) on the rat CaV3.1 channel and conducted structure-activity relationship studies by characterizing the interaction of 119 purpurealidin analogs. Next, the mechanism of action of the four most potent analogs was investigated. Analogs 74, 76, 79, and 99 showed a potent inhibition on the CaV3.1 channel with IC50's at approximately 3 µM. No shift of the activation curve could be observed, suggesting that these compounds act like a pore blocker obstructing the ion flow by binding in the pore region of the CaV3.1 channel. A selectivity screening showed that these analogs are also active on hERG channels. Collectively, a new class of CaV3 channel inhibitors has been discovered and the structure-function studies provide new insights into the synthetic design of drugs and the mechanism of interaction with T-type CaV channels.

Design and characterization of a novel structural class of Kv1.3 inhibitors.

The voltage-gated potassium channel Kv1.3 is involved in multiple autoimmune diseases, such as multiple sclerosis, rheumatoid arthritis, diabetes mellitus type 1 and psoriasis. In many auto-immune diseases better treatment options are desired as existing therapies are often ineffective or become less effective over time, for which Kv1.3 inhibitors arise as promising candidates. In this study, five compounds were selected based on a 3D similarity searching methodology and subsequently screened ex vivo on the Kv1.3 channel. The screening resulted in two compounds inhibiting the Kv1.3 channel, of which TVS-12 was the most potent compound, while TVS-06 -although less potent- showed an excellent selectivity for Kv1.3. For both compounds the mechanism of action was investigated by an electrophysiological characterization on the Kv1.3 channel and three Kv1.3 mutants, designed to resemble the pore region of Kv1.2 channels. Structurally, the presence of a benzene ring and/or an oxane ring seems to cause a better interaction with the Kv1.3 channel, resulting in a 20-fold higher potency for TVS-12.

Design, synthesis and biological evaluation of novel DNA gyrase inhibitors and their siderophore mimic conjugates.

Bacterial DNA gyrase is an important target for the development of novel antibacterial drugs, which are urgently needed because of high level of antibiotic resistance worldwide. We designed and synthesized new 4,5,6,7-tetrahydrobenzo[d]thiazole-based DNA gyrase B inhibitors and their conjugates with siderophore mimics, which were introduced to increase the uptake of inhibitors into the bacterial cytoplasm. The most potent conjugate 34 had an IC50 of 58 nM against Escherichia coli DNA gyrase and displayed MIC of 14 µg/mL against E. coli ΔtolC strain. Only minor improvements in the antibacterial activities against wild-type E. coli in low-iron conditions were seen for DNA gyrase inhibitor - siderophore mimic conjugates.

Antimicrobial activity of the marine alkaloids, clathrodin and oroidin, and their synthetic analogues.

Marine organisms produce secondary metabolites that may be valuable for the development of novel drug leads as such and can also provide structural scaffolds for the design and synthesis of novel bioactive compounds. The marine alkaloids, clathrodin and oroidin, which were originally isolated from sponges of the genus, Agelas, were prepared and evaluated for their antimicrobial activity against three bacterial strains (Enterococcus faecalis, Staphylococcus aureus and Escherichia coli) and one fungal strain (Candida albicans), and oroidin was found to possess promising Gram-positive antibacterial activity. Using oroidin as a scaffold, 34 new analogues were designed, prepared and screened for their antimicrobial properties. Of these compounds, 12 exhibited >80% inhibition of the growth of at least one microorganism at a concentration of 50 µM. The most active derivative was found to be 4-phenyl-2-aminoimidazole 6h, which exhibited MIC90 (minimum inhibitory concentration) values of 12.5 µM against the Gram-positive bacteria and 50 µM against E. coli. The selectivity index between S. aureus and mammalian cells, which is important to consider in the evaluation of a compound's potential as an antimicrobial lead, was found to be 2.9 for compound 6h.

Evaluation of Michael-type acceptor reactivity of 5-benzylidenebarbiturates, 5-benzylidenerhodanines, and related heterocycles using NMR.

Despite existing experimental and computational tools to assess the risk, the non-specific chemical modification of protein thiol groups remains a significant source of false-positive hits, particularly in academic drug discovery. Herein, we describe the application of a simple NMR method in a systematic study on the reactivity of 5-benzylidenebarbiturates, 5-benzylidenerhodanines, and their related oxo-heterocycles, which have been associated with numerous biological activities and have recently gained a reputation as unselective promiscuous binders. Using this method, we confirmed the reactivity of 5-benzylidenebarbiturates, which are known to easily form Michael adducts with nucleophiles. In contrast, 5-benzylidene five-membered oxo-heterocycles revealed almost insignificant reactivity. We can conclude that the distinct binding profile of the most controversial compounds, 5-benzylidenerhodanines, is not necessarily related to their unspecific Michael acceptor reactivity.

Benzothiazole DNA gyrase inhibitors and their conjugates with siderophore mimics: design, synthesis and evaluation.

Benzothiazole-based bacterial DNA gyrase and topoisomerase IV inhibitors are promising new antibacterial agents with potent activity against Gram-positive and Gram-negative bacterial strains. The aim of this study was to improve the uptake of these inhibitors into the cytoplasm of Gram-negative bacteria by conjugating them to the small siderophore mimics. The best conjugate 18b displayed potent Escherichia coli DNA gyrase and topoisomerase IV inhibition. The interaction analysis of molecular dynamics simulation trajectory showed the important contribution of the siderophore mimic moiety to binding affinity. By NMR spectroscopy, we demonstrated that the hydroxypyridinone moiety alone was responsible for the chelation of iron(iii). Moreover, 18b showed an enhancement of antibacterial activity against E. coli JW5503 in an iron-depleted medium, clearly indicating an increased uptake of 18b in this bacterial strain.

Estimation of passive gastrointestinal absorption of new dual DNA gyrase and topoisomerase IV inhibitors using PAMPA and biopartitioning micellar chromatography and quantitative structure-retention relationship analysis.

DNA gyrase and topoisomerase IV play significant role in maintaining the correct structure of DNA during replication and they have been identified as validated targets in antibacterial drug discovery. Inadequate pharmacokinetic properties are responsible for many failures during drug discovery and their estimation in the early phase of this process maximizes the chance of getting useful drug candidates. Passive gastrointestinal absorption of a selected group of thirteen dual DNA gyrase and topoisomerase IV inhibitors was estimated using two in vitro tests - parallel artificial membrane permeability assay (PAMPA) and biopartitioning micellar chromatography (BMC). Due to good correlation between obtained results, passive gastrointestinal absorption of remaining ten compounds was estimated using only BMC. With this experimental setup, it was possible to identify compounds with high values of retention factors (k) and highest expected passive gastrointestinal absorption, and compounds with low values of k for which low passive gastrointestinal absorption is predicted. Quantitative structure-retention relationship (QSRR) modelling was performed by creating multiple linear regression (MLR), partial least squares (PLS) and support vector machines (SVM) models. Descriptors with the highest influence on retention factor were identified and their interpretation can be used for the design of new compounds with improved passive gastrointestinal absorption.

Exploring the 5-Substituted 2-Aminobenzothiazole-Based DNA Gyrase B Inhibitors Active against ESKAPE Pathogens.

We present a new series of 2-aminobenzothiazole-based DNA gyrase B inhibitors with promising activity against ESKAPE bacterial pathogens. Based on the binding information extracted from the cocrystal structure of DNA gyrase B inhibitor A, in complex with Escherichia coli GyrB24, we expanded the chemical space of the benzothiazole-based series to the C5 position of the benzothiazole ring. In particular, compound E showed low nanomolar inhibition of DNA gyrase (IC50 < 10 nM) and broad-spectrum antibacterial activity against pathogens belonging to the ESKAPE group, with the minimum inhibitory concentration < 0.03 µg/mL for most Gram-positive strains and 4-16 µg/mL against Gram-negative E. coli, Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae. To understand the binding mode of the synthesized inhibitors, a combination of docking calculations, molecular dynamics (MD) simulations, and MD-derived structure-based pharmacophore modeling was performed. The computational analysis has revealed that the substitution at position C5 can be used to modify the physicochemical properties and antibacterial spectrum and enhance the inhibitory potency of the compounds. Additionally, a discussion of challenges associated with the synthesis of 5-substituted 2-aminobenzothiazoles is presented.

Garbage in, garbage out: how reliable training data improved a virtual screening approach against SARS-CoV-2 MPro.

Introduction: The identification of chemical compounds that interfere with SARS-CoV-2 replication continues to be a priority in several academic and pharmaceutical laboratories. Computational tools and approaches have the power to integrate, process and analyze multiple data in a short time. However, these initiatives may yield unrealistic results if the applied models are not inferred from reliable data and the resulting predictions are not confirmed by experimental evidence. Methods: We undertook a drug discovery campaign against the essential major protease (MPro) from SARS-CoV-2, which relied on an in silico search strategy -performed in a large and diverse chemolibrary- complemented by experimental validation. The computational method comprises a recently reported ligand-based approach developed upon refinement/learning cycles, and structure-based approximations. Search models were applied to both retrospective (in silico) and prospective (experimentally confirmed) screening. Results: The first generation of ligand-based models were fed by data, which to a great extent, had not been published in peer-reviewed articles. The first screening campaign performed with 188 compounds (46 in silico hits and 100 analogues, and 40 unrelated compounds: flavonols and pyrazoles) yielded three hits against MPro (IC50 ≤ 25 µM): two analogues of in silico hits (one glycoside and one benzo-thiazol) and one flavonol. A second generation of ligand-based models was developed based on this negative information and newly published peer-reviewed data for MPro inhibitors. This led to 43 new hit candidates belonging to different chemical families. From 45 compounds (28 in silico hits and 17 related analogues) tested in the second screening campaign, eight inhibited MPro with IC50 = 0.12-20 µM and five of them also impaired the proliferation of SARS-CoV-2 in Vero cells (EC50 7-45 µM). Discussion: Our study provides an example of a virtuous loop between computational and experimental approaches applied to target-focused drug discovery against a major and global pathogen, reaffirming the well-known "garbage in, garbage out" machine learning principle.

New bisphenol A and bisphenol S analogs: Evaluation of their hERα agonistic and antagonistic activities using the OECD 455 in-vitro assay and molecular modeling.

Bisphenol A (BPA) and bisphenol S (BPS) are agonists of hERα receptors and due to BPA regulations in many countries, several substitutes that are close analogs to BPA and BPS were developed. In the presented study, we have determined human estrogen receptor (hER)α agonist and antagonist activities with the validated OECD assay with the hERα-Hela9903 cell line for five different chemical classes of BPA and BPS analogs. This study also defined clear structure-activity relationships for agonist and antagonist activities of the 12 bisphenols on hERα, which are supported by molecular docking studies. These data show that classical analogs of BPA (e.g., bisphenols B, C, AP, E) have comparable or superior estrogenic agonist potencies compared to BPA and BPS. The most potent of these hERα agonists were even more potent than BPA, as bisphenol B and C, with IC50 values of 0.31 µM and 0.48 µM, respectively. Among these selected bisphenols, 4-4'-methylenebis (oxyethylenethio)diphenol was the most potent hERα antagonist, with an IC50 of 0.39 µM. The estrogenic agonist and antagonist potencies of these different chemical classes of BPA and BPS analogs are mutually comparable and can be used as a basis for further structure-activity relationships studies and human risk assessment.

Design of New Potent and Selective Thiophene-Based KV1.3 Inhibitors and Their Potential for Anticancer Activity.

The voltage-gated potassium channel KV1.3 has been recognized as a tumor marker and represents a promising new target for the discovery of new anticancer drugs. We designed a novel structural class of KV1.3 inhibitors through structural optimization of benzamide-based hit compounds and structure-activity relationship studies. The potency and selectivity of the new KV1.3 inhibitors were investigated using whole-cell patch- and voltage-clamp experiments. 2D and 3D cell models were used to determine antiproliferative activity. Structural optimization resulted in the most potent and selective KV1.3 inhibitor 44 in the series with an IC50 value of 470 nM in oocytes and 950 nM in Ltk- cells. KV1.3 inhibitor 4 induced significant apoptosis in Colo-357 spheroids, while 14, 37, 43, and 44 significantly inhibited Panc-1 proliferation.

Structure-Activity Relationships of Benzothiazole-Based Hsp90 C-Terminal-Domain Inhibitors.

Heat shock protein 90 (Hsp90) is a chaperone responsible for the maturation of many cancer-related proteins, and is therefore an important target for the design of new anticancer agents. Several Hsp90 N-terminal domain inhibitors have been evaluated in clinical trials, but none have been approved as cancer therapies. This is partly due to induction of the heat shock response, which can be avoided using Hsp90 C-terminal-domain (CTD) inhibition. Several structural features have been shown to be useful in the design of Hsp90 CTD inhibitors, including an aromatic ring, a cationic center and the benzothiazole moiety. This study established a previously unknown link between these structural motifs. Using ligand-based design methodologies and structure-based pharmacophore models, a library of 29 benzothiazole-based Hsp90 CTD inhibitors was prepared, and their antiproliferative activities were evaluated in MCF-7 breast cancer cells. Several showed low-micromolar IC50, with the most potent being compounds 5g and 9i (IC50, 2.8 ± 0.1, 3.9 ± 0.1 µM, respectively). Based on these results, a ligand-based structure-activity relationship model was built, and molecular dynamics simulation was performed to elaborate the binding mode of compound 9i. Moreover, compound 9i showed degradation of Hsp90 client proteins and no induction of the heat shock response.

3D Pharmacophore-Based Discovery of Novel KV10.1 Inhibitors with Antiproliferative Activity.

(1) Background: The voltage-gated potassium channel KV10.1 (Eag1) is considered a near- universal tumour marker and represents a promising new target for the discovery of novel anticancer drugs. (2) Methods: We utilized the ligand-based drug discovery methodology using 3D pharmacophore modelling and medicinal chemistry approaches to prepare a novel structural class of KV10.1 inhibitors. Whole-cell patch clamp experiments were used to investigate potency, selectivity, kinetics and mode of inhibition. Anticancer activity was determined using 2D and 3D cell-based models. (3) Results: The virtual screening hit compound ZVS-08 discovered by 3D pharmacophore modelling exhibited an IC50 value of 3.70 µM against KV10.1 and inhibited the channel in a voltage-dependent manner consistent with the action of a gating modifier. Structural optimization resulted in the most potent KV10.1 inhibitor of the series with an IC50 value of 740 nM, which was potent on the MCF-7 cell line expressing high KV10.1 levels and low hERG levels, induced significant apoptosis in tumour spheroids of Colo-357 cells and was not mutagenic. (4) Conclusions: Computational ligand-based drug design methods can be successful in the discovery of new potent KV10.1 inhibitors. The main problem in the field of KV10.1 inhibitors remains selectivity against the hERG channel, which needs to be addressed in the future also with target-based drug design methods.

Practical Synthesis and Application of Halogen-Doped Pyrrole Building Blocks.

A practical access to four new halogen-substituted pyrrole building blocks was realized in two to five synthetic steps from commercially available starting materials. The target compounds were prepared on a 50 mg to 1 g scale, and their conversion to nanomolar inhibitors of bacterial DNA gyrase B was demonstrated for three of the prepared building blocks to showcase the usefulness of such chemical motifs in medicinal chemistry.

New dual ATP-competitive inhibitors of bacterial DNA gyrase and topoisomerase IV active against ESKAPE pathogens.

The rise in multidrug-resistant bacteria defines the need for identification of new antibacterial agents that are less prone to resistance acquisition. Compounds that simultaneously inhibit multiple bacterial targets are more likely to suppress the evolution of target-based resistance than monotargeting compounds. The structurally similar ATP binding sites of DNA gyrase and topoisomerase Ⅳ offer an opportunity to accomplish this goal. Here we present the design and structure-activity relationship analysis of balanced, low nanomolar inhibitors of bacterial DNA gyrase and topoisomerase IV that show potent antibacterial activities against the ESKAPE pathogens. For inhibitor 31c, a crystal structure in complex with Staphylococcus aureus DNA gyrase B was obtained that confirms the mode of action of these compounds. The best inhibitor, 31h, does not show any in vitro cytotoxicity and has excellent potency against Gram-positive (MICs: range, 0.0078-0.0625 µg/mL) and Gram-negative pathogens (MICs: range, 1-2 µg/mL). Furthermore, 31h inhibits GyrB mutants that can develop resistance to other drugs. Based on these data, we expect that structural derivatives of 31h will represent a step toward clinically efficacious multitargeting antimicrobials that are not impacted by existing antimicrobial resistance.

In silico discovery of 2-amino-4-(2,4-dihydroxyphenyl)thiazoles as novel inhibitors of DNA gyrase B.

Cyclothialidines are a class of bacterial DNA gyrase B (GyrB) subunit inhibitors, targeting its ATP-binding site. Starting from the available structural information on cyclothialidine GR122222X (2), an in silico virtual screening campaign was designed combining molecular docking calculations with three-dimensional structure-based pharmacophore information. A novel class of 2-amino-4-(2,4-dihydroxyphenyl)thiazole based inhibitors (5-9) with low micromolar antigyrase activity was discovered.

In vitro estrogenic activity of binary and multicomponent mixtures with bisphenol A.

Bisphenol A and its analogs are environmental contaminants with well known estrogenic and anti-androgenic activities. In studies of human biomonitoring, simultaneous exposure to multiple bisphenols was shown in different biological samples, at picomolar to low nanomolar concentrations. Evaluation of their combined toxicities will therefore be a more realistic and reliable predictor for estimation of health risks than evaluation of only the single chemicals. In the present study, estrogenic activities of individual bisphenols were evaluated, along with their binary and multicomponent mixtures including three- and four-component mixtures, using the Organisation for Economic Co-operation and Development validated transactivation assay with the hERα-Hela9903 cell line. Concentration-dependent estrogenic activity was confirmed for all of the tested bisphenols, in the nanomolar to micromolar range. Estrogenic activities of binary and multicomponent mixtures followed a concentration addition model. Although exposure to individual bisphenols remains below their effective doses, we demonstrate that as a mixture, they can contribute additively to toxicity. This study thus emphasizes the importance of mixture toxicity evaluation for risk assessment of compounds that act like the bisphenols.

Efficient Synthesis of Hydroxy-Substituted 2-Aminobenzo[d]thiazole-6-carboxylic Acid Derivatives as New Building Blocks in Drug Discovery.

Benzo[d]thiazole is widely used in synthetic and medicinal chemistry, and it is a component of many compounds and drugs that have several different bioactivities. Herein, we describe an elegant pathway for synthesis of methyl 4- and 5-hydroxy-2-amino-benzo[d]thiazole-6-carboxylates as building blocks that can be substituted at four different positions on the bicycle and thus offer the possibility to thoroughly explore the chemical space around the molecule studied as a ligand for the chosen target. A series of 12 new compounds was prepared using the described methods and Williamson ether synthesis.