Antibiotic class with potent in vivo activity targeting lipopolysaccharide synthesis in Gram-negative bacteria.

Here, we describe the identification of an antibiotic class acting via LpxH, a clinically unexploited target in lipopolysaccharide synthesis. The lipopolysaccharide synthesis pathway is essential in most Gram-negative bacteria and there is no analogous pathway in humans. Based on a series of phenotypic screens, we identified a hit targeting this pathway that had activity on efflux-defective strains of Escherichia coli. We recognized common structural elements between this hit and a previously published inhibitor, also with activity against efflux-deficient bacteria. With the help of X-ray structures, this information was used to design inhibitors with activity on efflux-proficient, wild-type strains. Optimization of properties such as solubility, metabolic stability and serum protein binding resulted in compounds having potent in vivo efficacy against bloodstream infections caused by the critical Gram-negative pathogens E. coli and Klebsiella pneumoniae. Other favorable properties of the series include a lack of pre-existing resistance in clinical isolates, and no loss of activity against strains expressing extended-spectrum-ß-lactamase, metallo-ß-lactamase, or carbapenemase-resistance genes. Further development of this class of antibiotics could make an important contribution to the ongoing struggle against antibiotic resistance.

Bacterial type I signal peptidase inhibitors - Optimized hits from nature.

The ever-increasing number of bacteria resistant to the currently available antibacterial agents is a great medical problem today, and new antibiotics with novel mechanisms of action are urgently needed. Among the validated antibacterial drug targets against which new classes of antibiotics might be directed is bacterial type I signal peptidase (SPase I), an essential part of the Tat and Sec secretory systems. SPase I is responsible for the hydrolysis of the N-terminal signal peptides from proteins secreted across the cytoplasmic membrane and plays a key role in bacterial viability and virulence. This review focuses on the antibacterial activity of natural and synthetic SPase I inhibitors reported to date, namely ß-lactams, lipopeptides, and arylomycins, but also an example of SPase I activator was presented.

Novel Allosteric Mechanism of Dual p53/MDM2 and p53/MDM4 Inhibition by a Small Molecule.

Restoration of the p53 tumor suppressor for personalised cancer therapy is a promising treatment strategy. However, several high-affinity MDM2 inhibitors have shown substantial side effects in clinical trials. Thus, elucidation of the molecular mechanisms of action of p53 reactivating molecules with alternative functional principle is of the utmost importance. Here, we report a discovery of a novel allosteric mechanism of p53 reactivation through targeting the p53 N-terminus which promotes inhibition of both p53/MDM2 (murine double minute 2) and p53/MDM4 interactions. Using biochemical assays and molecular docking, we identified the binding site of two p53 reactivating molecules, RITA (reactivation of p53 and induction of tumor cell apoptosis) and protoporphyrin IX (PpIX). Ion mobility-mass spectrometry revealed that the binding of RITA to serine 33 and serine 37 is responsible for inducing the allosteric shift in p53, which shields the MDM2 binding residues of p53 and prevents its interactions with MDM2 and MDM4. Our results point to an alternative mechanism of blocking p53 interaction with MDM2 and MDM4 and may pave the way for the development of novel allosteric inhibitors of p53/MDM2 and p53/MDM4 interactions.

Synthesis and In Vitro Biological Evaluation of Quinolinyl Pyrimidines Targeting Type II NADH-Dehydrogenase (NDH-2).

Type II NADH dehydrogenase (NDH-2) is an essential component of electron transfer in many microbial pathogens but has remained largely unexplored as a potential drug target. Previously, quinolinyl pyrimidines were shown to inhibit Mycobacterium tuberculosis NDH-2, as well as the growth of the bacteria [Shirude, P. S.; ACS Med. Chem. Lett. 2012, 3, 736-740]. Here, we synthesized a number of novel quinolinyl pyrimidines and investigated their properties. In terms of inhibition of the NDH-2 enzymes from M. tuberculosis and Mycobacterium smegmatis, the best compounds were of similar potency to previously reported inhibitors of the same class (half-maximal inhibitory concentration (IC50) values in the low-µM range). However, a number of the compounds had much better activity against Gram-negative pathogens, with minimum inhibitory concentrations (MICs) as low as 2 µg/mL. Multivariate analyses (partial least-squares (PLS) and principle component analysis (PCA)) showed that overall ligand charge was one of the most important factors in determining antibacterial activity, with patterns that varied depending on the particular bacterial species. In some cases (e.g., mycobacteria), there was a clear correlation between the IC50 values and the observed MICs, while in other instances, no such correlation was evident. When tested against a panel of protozoan parasites, the compounds failed to show activity that was not linked to cytotoxicity. Further, a strong correlation between hydrophobicity (estimated as clog P) and cytotoxicity was revealed; more hydrophobic analogues were more cytotoxic. By contrast, antibacterial MIC values and cytotoxicity were not well correlated, suggesting that the quinolinyl pyrimidines can be optimized further as antimicrobial agents.

Computational studies of molecular pre-organization through macrocyclization: Conformational distribution analysis of closely related non-macrocyclic and macrocyclic analogs.

Macrocycles form an important compound class in medicinal chemistry due to their interesting structural and biological properties. To help design macrocycles, it is important to understand how the conformational preferences are affected upon macrocyclization of a lead compound. To address this, we collected a unique data set of protein-ligand complexes containing "non-macrocyclic" ("linear") ligands matched with macrocyclic analogs binding to the same protein in a similar pose. Out of the 39 co-crystallized ligands considered, 10 were linear and 29 were macrocyclic. To enable a more general analysis, 128 additional ligands from the publications associated with these protein data bank entries were added to the data set. Using in total 167 collected ligands, we investigated if the conformers in the macrocyclic conformational ensembles were more similar to the bioactive conformation in comparison to the conformers of their linear counterparts. Unexpectedly, in most cases the macrocycle conformational ensemble distributions were not very different from those of the linear compounds. Thus, care should be taken when designing macrocycles with the aim to focus their conformational preference towards the bioactive conformation. We also set out to investigate potential conformational flexibility differences between the two compound classes, computational energy window settings and evaluate a literature metric for approximating the conformational focusing on the bioactive conformation.

Antibacterial sulfonimidamide-based oligopeptides as type I signal peptidase inhibitors: Synthesis and biological evaluation.

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.

Towards the sustainable discovery and development of new antibiotics.

An ever-increasing demand for novel antimicrobials to treat life-threatening infections caused by the global spread of multidrug-resistant bacterial pathogens stands in stark contrast to the current level of investment in their development, particularly in the fields of natural-product-derived and synthetic small molecules. New agents displaying innovative chemistry and modes of action are desperately needed worldwide to tackle the public health menace posed by antimicrobial resistance. Here, our consortium presents a strategic blueprint to substantially improve our ability to discover and develop new antibiotics. We propose both short-term and long-term solutions to overcome the most urgent limitations in the various sectors of research and funding, aiming to bridge the gap between academic, industrial and political stakeholders, and to unite interdisciplinary expertise in order to efficiently fuel the translational pipeline for the benefit of future generations.

Towards the sustainable discovery and development of new antibiotics.

An ever-increasing demand for novel antimicrobials to treat life-threatening infections caused by the global spread of multidrug-resistant bacterial pathogens stands in stark contrast to the current level of investment in their development, particularly in the fields of natural-product-derived and synthetic small molecules. New agents displaying innovative chemistry and modes of action are desperately needed worldwide to tackle the public health menace posed by antimicrobial resistance. Here, our consortium presents a strategic blueprint to substantially improve our ability to discover and develop new antibiotics. We propose both short-term and long-term solutions to overcome the most urgent limitations in the various sectors of research and funding, aiming to bridge the gap between academic, industrial and political stakeholders, and to unite interdisciplinary expertise in order to efficiently fuel the translational pipeline for the benefit of future generations.

Conformational analysis of macrocycles: comparing general and specialized methods.

Macrocycles represent an important class of medicinally relevant small molecules due to their interesting biological properties. Therefore, a firm understanding of their conformational preferences is important for drug design. Given the importance of macrocycle-protein modelling in drug discovery, we envisaged that a systematic study of both classical and recent specialized methods would provide guidance for other practitioners within the field. In this study we compare the performance of the general, well established conformational analysis methods Monte Carlo Multiple Minimum (MCMM) and Mixed Torsional/Low-Mode sampling (MTLMOD) with two more recent and specialized macrocycle sampling techniques: MacroModel macrocycle Baseline Search (MD/LLMOD) and Prime macrocycle conformational sampling (PRIME-MCS). Using macrocycles extracted from 44 macrocycle-protein X-ray crystallography complexes, we evaluated each method based on their ability to (i) generate unique conformers, (ii) generate unique macrocycle ring conformations, (iii) identify the global energy minimum, (iv) identify conformers similar to the X-ray ligand conformation after Protein Preparation Wizard treatment (X-rayppw), and (v) to the X-rayppw ring conformation. Computational speed was also considered. In addition, conformational coverage, as defined by the number of conformations identified, was studied. In order to study the relative energies of the bioactive conformations, the energy differences between the global energy minima and the energy minimized X-rayppw structures and, the global energy minima and the MCMM-Exhaustive (1,000,000 search steps) generated conformers closest to the X-rayppw structure, were calculated and analysed. All searches were performed using relatively short run times (10,000 steps for MCMM, MTLMOD and MD/LLMOD). To assess the performance of the methods, they were compared to an exhaustive MCMM search using 1,000,000 search steps for each of the 44 macrocycles (requiring ca 200 times more CPU time). Prior to our analysis, we also investigated if the general search methods MCMM and MTLMOD could also be optimized for macrocycle conformational sampling. Taken together, our work concludes that the more general methods can be optimized for macrocycle modelling by slightly adjusting the settings around the ring closure bond. In most cases, MCMM and MTLMOD with either standard or enhanced settings performed well in comparison to the more specialized macrocycle sampling methods MD/LLMOD and PRIME-MCS. When using enhanced settings for MCMM and MTLMOD, the X-rayppw conformation was regenerated with the greatest accuracy. The, MD/LLMOD emerged as the most efficient method for generating the global energy minima.

The global preclinical antibacterial pipeline.

Antibacterial resistance is a great concern and requires global action. A critical question is whether enough new antibacterial drugs are being discovered and developed. A review of the clinical antibacterial drug pipeline was recently published, but comprehensive information about the global preclinical pipeline is unavailable. This Review focuses on discovery and preclinical development projects and has found, as of 1 May 2019, 407 antibacterial projects from 314 institutions. The focus is on Gram-negative pathogens, particularly bacteria on the WHO priority bacteria list. The preclinical pipeline is characterized by high levels of diversity and interesting scientific concepts, with 135 projects on direct-acting small molecules that represent new classes, new targets or new mechanisms of action. There is also a strong trend towards non-traditional approaches, including diverse antivirulence approaches, microbiome-modifying strategies, and engineered phages and probiotics. The high number of pathogen-specific and adjunctive approaches is unprecedented in antibiotic history. Translational hurdles are not adequately addressed yet, especially development pathways to show clinical impact of non-traditional approaches. The innovative potential of the preclinical pipeline compared with the clinical pipeline is encouraging but fragile. Much more work, focus and funding are needed for the novel approaches to result in effective antibacterial therapies to sustainably combat antibacterial resistance.

Boronic ester-linked macrocyclic lipopeptides as serine protease inhibitors targeting Escherichia coli type I signal peptidase.

Type I signal peptidase, with its vital role in bacterial viability, is a promising but underexploited antibacterial drug target. In the light of steadily increasing rates of antimicrobial resistance, we have developed novel macrocyclic lipopeptides, linking P2 and P1' by a boronic ester warhead, capable of inhibiting Escherichia coli type I signal peptidase (EcLepB) and exhibiting good antibacterial activity. Structural modifications of the macrocyclic ring, the peptide sequence and the lipophilic tail led us to 14 novel macrocyclic boronic esters. It could be shown that macrocyclization is well tolerated in terms of EcLepB inhibition and antibacterial activity. Among the synthesized macrocycles, potent enzyme inhibitors in the low nanomolar range (e.g. compound 42f, EcLepB IC50 = 29 nM) were identified also showing good antimicrobial activity (e.g. compound 42b, E. coli WT MIC = 16 µg/mL). The unique macrocyclic boronic esters described here were based on previously published linear lipopeptidic EcLepB inhibitors in an attempt to address cytotoxicity and hemolysis. We show herein that structural changes to the macrocyclic ring influence both the cytotoxicity and hemolytic activity suggesting that the P2 to P1' linker provide means for optimizing off-target effects. However, for the present set of compounds we were not able to separate the antibacterial activity and cytotoxic effect.

Predicting the Rate of Skin Penetration Using an Aggregated Conformal Prediction Framework.

Skin serves as a drug administration route, and skin permeability of chemicals is of significant interest in the pharmaceutical and cosmetic industries. An aggregated conformal prediction (ACP) framework was used to build models for predicting the permeation rate (log Kp) of chemical compounds through human skin. The conformal prediction method gives as an output the prediction range at a given level of confidence for each compound, which enables the user to make a more informed decision when, for example, suggesting the next compound to prepare. Predictive models were built using both the random forest and the support vector machine methods and were based on experimentally derived permeability data on 211 diverse compounds. The derived models were of similar predictive quality as compared to earlier published models but have the extra advantage of not only presenting a single predicted value for each compound but also a reliable, individually assigned prediction range. The models use calculated descriptors and can quickly predict the skin permeation rate of new compounds.

Docking of Macrocycles: Comparing Rigid and Flexible Docking in Glide.

In recent years, there has been an increased interest in using macrocyclic compounds for drug discovery and development. For docking of these commonly large and flexible compounds to be addressed, a screening and a validation set were assembled from the PDB consisting of 16 and 31 macrocycle-containing protein complexes, respectively. The macrocycles were docked in Glide by rigid docking of pregenerated conformational ensembles produced by the macrocycle conformational sampling method (MCS) in Schrödinger Release 2015-3 or by direct Glide flexible docking after performing ring-templating. The two protocols were compared to rigid docking of pregenerated conformational ensembles produced by an exhaustive Monte Carlo multiple minimum (MCMM) conformational search and a shorter MCMM conformational search (MCMM-short). The docking accuracy was evaluated and expressed as the RMSD between the heavy atoms of the ligand as found in the X-ray structure after refinement and the poses obtained by the docking protocols. The median RMSD values for top-scored poses of the screening set were 0.83, 0.80, 0.88, and 0.58 Å for MCMM, MCMM-short, MCS, and Glide flexible docking, respectively. There was no statistically significant difference in the performance between rigid docking of pregenerated conformations produced by the MCS and direct docking using Glide flexible docking. However, the flexible docking protocol was 2-times faster in docking the screening set compared to that of the MCS protocol. In a final study, the new Prime-MCS method was evaluated in Schrödinger Release 2016-3. This method is faster compared that of to MCS; however, the conformations generated were found to be suboptimal for rigid docking. Therefore, on the basis of timing, accuracy, and ease of set up, standard Glide flexible docking with prior ring-templating is recommended over current gold standard protocols using rigid docking of pregenerated conformational ensembles.

Design, synthesis and in vitro biological evaluation of oligopeptides targeting E. coli type I signal peptidase (LepB).

Type I signal peptidases are potential targets for the development of new antibacterial agents. Here we report finding potent inhibitors of E. coli type I signal peptidase (LepB), by optimizing a previously reported hit compound, decanoyl-PTANA-CHO, through modifications at the N- and C-termini. Good improvements of inhibitory potency were obtained, with IC50s in the low nanomolar range. The best inhibitors also showed good antimicrobial activity, with MICs in the low µg/mL range for several bacterial species. The selection of resistant mutants provided strong support for LepB as the target of these compounds. The cytotoxicity and hemolytic profiles of these compounds are not optimal but the finding that minor structural changes cause the large effects on these properties suggests that there is potential for optimization in future studies.

Optimization and Evaluation of 5-Styryl-Oxathiazol-2-one Mycobacterium tuberculosis Proteasome Inhibitors as Potential Antitubercular Agents.

This is the first report of 5-styryl-oxathiazol-2-ones as inhibitors of the Mycobacterium tuberculosis (Mtb) proteasome. As part of the study, the structure-activity relationship of oxathiazolones as Mtb proteasome inhibitors has been investigated. Furthermore, the prepared compounds displayed a good selectivity profile for Mtb compared to the human proteasome. The 5-styryl-oxathiazol-2-one inhibitors identified showed little activity against replicating Mtb, but were rapidly bactericidal against nonreplicating bacteria. (E)-5-(4-Chlorostyryl)-1,3,4-oxathiazol-2-one) was most effective, reducing the colony-forming units (CFU)/mL below the detection limit in only seven days at all concentrations tested. The results suggest that this new class of Mtb proteasome inhibitors has the potential to be further developed into novel antitubercular agents for synergistic combination therapies with existing drugs.

Interconversion of Functional Activity by Minor Structural Alterations in Nonpeptide AT2 Receptor Ligands.

Migration of the methylene imidazole side chain in the first reported selective drug-like AT2 receptor agonist C21/M024 (1) delivered the AT2 receptor antagonist C38/M132 (2). We now report that the AT2 receptor antagonist compound 4, a biphenyl derivative that is structurally related to 2, is transformed to the agonist 6 by migration of the isobutyl group. The importance of the relative position of the methylene imidazole and the isobutyl substituent is highlighted herein.

Phototriggerable peptidomimetics for the inhibition of Mycobacterium tuberculosis ribonucleotide reductase by targeting protein-protein binding.

Incorporation of an artificial amino acid 2 with a stilbene chromophore into peptidomimetics with three to nine amino acids yields phototriggerable candidates for inhibition of the binding between the R1 and R2 subunits of the M. tuberculosis ribonucleotide reductase (RNR). Interstrand hydrogen bond probability was used as a guideline for predicting conformational preferences of the photoisomers. Binding of these inhibitors has been rationalized by docking studies with the R1 unit. Significant differences in binding of the photoisomers were observed. For the shorter peptidomimetics, stronger binding of the Z isomer might indicate hydrophobic interactions between the stilbene chromophore and the binding site.

Toward a benchmarking data set able to evaluate ligand- and structure-based virtual screening using public HTS data.

Virtual screening has the potential to accelerate and reduce costs of probe development and drug discovery. To develop and benchmark virtual screening methods, validation data sets are commonly used. Over the years, such data sets have been constructed to overcome the problems of analogue bias and artificial enrichment. With the rapid growth of public domain databases containing high-throughput screening data, such as the PubChem BioAssay database, there is an increased possibility to use such data for validation. In this study, we identify PubChem data sets suitable for validation of both structure- and ligand-based virtual screening methods. To achieve this, high-throughput screening data for which a crystal structure of the bioassay target was available in the PDB were identified. Thereafter, the data sets were inspected to identify structures and data suitable for use in validation studies. In this work, we present seven data sets (MMP13, DUSP3, PTPN22, EPHX2, CTDSP1, MAPK10, and CDK5) compiled using this method. In the seven data sets, the number of active compounds varies between 19 and 369 and the number of inactive compounds between 59 405 and 337 634. This gives a higher ratio of the number of inactive to active compounds than what is found in most benchmark data sets. We have also evaluated the screening performance using docking and 3D shape similarity with default settings. To characterize the data sets, we used physicochemical similarity and 2D fingerprint searches. We envision that these data sets can be a useful complement to current data sets used for method evaluation.

Vinylated linear P2 pyrimidinyloxyphenylglycine based inhibitors of the HCV NS3/4A protease and corresponding macrocycles.

With three recent market approvals and several inhibitors in advanced stages of development, the hepatitis C virus (HCV) NS3/4A protease represents a successful target for antiviral therapy against hepatitis C. As a consequence of dealing with viral diseases in general, there are concerns related to the emergence of drug resistant strains which calls for development of inhibitors with an alternative binding-mode than the existing highly optimized ones. We have previously reported on the use of phenylglycine as an alternative P2 residue in HCV NS3/4A protease inhibitors. Herein, we present the synthesis, structure-activity relationships and in vitro pharmacokinetic characterization of a diverse series of linear and macrocyclic P2 pyrimidinyloxyphenylglycine based inhibitors. With access to vinyl substituents in P3, P2 and P1' positions an initial probing of macrocyclization between different positions, using ring-closing metathesis (RCM) could be performed, after addressing some synthetic challenges. Biochemical results from the wild type enzyme and drug resistant variants (e.g., R155 K) indicate that P3-P1' macrocyclization, leaving the P2 substituent in a flexible mode, is a promising approach. Additionally, the study demonstrates that phenylglycine based inhibitors benefit from p-phenylpyrimidinyloxy and m-vinyl groups as well as from the combination with an aromatic P1 motif with alkenylic P1' elongations. In fact, linear P2-P1' spanning intermediate compounds based on these fragments were found to display promising inhibitory potencies and drug like properties.