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.

Western boundary currents regulated by interaction between ocean eddies and the atmosphere.

Current climate models systematically underestimate the strength of oceanic fronts associated with strong western boundary currents, such as the Kuroshio and Gulf Stream Extensions, and have difficulty simulating their positions at the mid-latitude ocean's western boundaries. Even with an enhanced grid resolution to resolve ocean mesoscale eddies-energetic circulations with horizontal scales of about a hundred kilometres that strongly interact with the fronts and currents-the bias problem can still persist; to improve climate models we need a better understanding of the dynamics governing these oceanic frontal regimes. Yet prevailing theories about the western boundary fronts are based on ocean internal dynamics without taking into consideration the intense air-sea feedbacks in these oceanic frontal regions. Here, by focusing on the Kuroshio Extension Jet east of Japan as the direct continuation of the Kuroshio, we show that feedback between ocean mesoscale eddies and the atmosphere (OME-A) is fundamental to the dynamics and control of these energetic currents. Suppressing OME-A feedback in eddy-resolving coupled climate model simulations results in a 20-40 per cent weakening in the Kuroshio Extension Jet. This is because OME-A feedback dominates eddy potential energy destruction, which dissipates more than 70 per cent of the eddy potential energy extracted from the Kuroshio Extension Jet. The absence of OME-A feedback inevitably leads to a reduction in eddy potential energy production in order to balance the energy budget, which results in a weakened mean current. The finding has important implications for improving climate models' representation of major oceanic fronts, which are essential components in the simulation and prediction of extratropical storms and other extreme events, as well as in the projection of the effect on these events of climate change.

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.

Design of Drug-Like Protein-Protein Interaction Stabilizers Guided By Chelation-Controlled Bioactive Conformation Stabilization.

Protein-protein interactions (PPIs) of 14-3-3 proteins are a model system for studying PPI stabilization. The complex natural product Fusicoccin A stabilizes many 14-3-3 PPIs but is not amenable for use in SAR studies, motivating the search for more drug-like chemical matter. However, drug-like 14-3-3 PPI stabilizers enabling such studies have remained elusive. An X-ray crystal structure of a PPI in complex with an extremely low potency stabilizer uncovered an unexpected non-protein interacting, ligand-chelated Mg2+ leading to the discovery of metal-ion-dependent 14-3-3 PPI stabilization potency. This originates from a novel chelation-controlled bioactive conformation stabilization effect. Metal chelation has been associated with pan-assay interference compounds (PAINS) and frequent hitter behavior, but chelation can evidently also lead to true potency gains and find use as a medicinal chemistry strategy to guide compound optimization. To demonstrate this, we exploited the effect to design the first potent, selective, and drug-like 14-3-3 PPI stabilizers.

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.

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.

Interannual atmospheric variability forced by the deep equatorial Atlantic Ocean.

Climate variability in the tropical Atlantic Ocean is determined by large-scale ocean-atmosphere interactions, which particularly affect deep atmospheric convection over the ocean and surrounding continents. Apart from influences from the Pacific El Niño/Southern Oscillation and the North Atlantic Oscillation, the tropical Atlantic variability is thought to be dominated by two distinct ocean-atmosphere coupled modes of variability that are characterized by meridional and zonal sea-surface-temperature gradients and are mainly active on decadal and interannual timescales, respectively. Here we report evidence that the intrinsic ocean dynamics of the deep equatorial Atlantic can also affect sea surface temperature, wind and rainfall in the tropical Atlantic region and constitutes a 4.5-yr climate cycle. Specifically, vertically alternating deep zonal jets of short vertical wavelength with a period of about 4.5 yr and amplitudes of more than 10 cm s(-1) are observed, in the deep Atlantic, to propagate their energy upwards, towards the surface. They are linked, at the sea surface, to equatorial zonal current anomalies and eastern Atlantic temperature anomalies that have amplitudes of about 6 cm s(-1) and 0.4 °C, respectively, and are associated with distinct wind and rainfall patterns. Although deep jets are also observed in the Pacific and Indian oceans, only the Atlantic deep jets seem to oscillate on interannual timescales. Our knowledge of the persistence and regularity of these jets is limited by the availability of high-quality data. Despite this caveat, the oscillatory behaviour can still be used to improve predictions of sea surface temperature in the tropical Atlantic. Deep-jet generation and upward energy transmission through the Equatorial Undercurrent warrant further theoretical study.

Synthesis of 11C-Labelled Ureas by Palladium(II)-Mediated Oxidative Carbonylation.

Positron emission tomography is an imaging technique with applications in clinical settings as well as in basic research for the study of biological processes. A PET tracer, a biologically active molecule where a positron-emitting radioisotope such as carbon-11 has been incorporated, is used for the studies. Development of robust methods for incorporation of the radioisotope is therefore of the utmost importance. The urea functional group is present in many biologically active compounds and is thus an attractive target for incorporation of carbon-11 in the form of [11C]carbon monoxide. Starting with amines and [11C]carbon monoxide, both symmetrical and unsymmetrical 11C-labelled ureas were synthesised via a palladium(II)-mediated oxidative carbonylation and obtained in decay-corrected radiochemical yields up to 65%. The added advantage of using [11C]carbon monoxide was shown by the molar activity obtained for an inhibitor of soluble epoxide hydrolase (247 GBq/µmol-319 GBq/µmol). DFT calculations were found to support a reaction mechanism proceeding through an 11C-labelled isocyanate intermediate.

Discovery of pyrazinone based compounds that potently inhibit the drug-resistant enzyme variant R155K of the hepatitis C virus NS3 protease.

Herein, we present the design and synthesis of 2(1H)-pyrazinone based HCV NS3 protease inhibitors with variations in the C-terminus. Biochemical evaluation was performed using genotype 1a, both the wild-type and the drug resistant enzyme variant, R155K. Surprisingly, compounds without an acidic sulfonamide retained good inhibition, challenging our previous molecular docking model. Moreover, selected compounds in this series showed nanomolar potency against R155K NS3 protease; which generally confer resistance to all HCV NS3 protease inhibitors approved or in clinical trials. These results further strengthen the potential of this novel substance class, being very different to the approved drugs and clinical candidates, in the development of inhibitors less sensitive to drug resistance.

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.

Coastal trapped waves and tidal mixing control primary production in the tropical Angolan upwelling system.

Eastern boundary upwelling systems are hotspots of marine life and primary production. The strength and seasonality of upwelling in these systems are usually related to local wind forcing. However, in some tropical upwelling systems, seasonal maxima of productivity occur when upwelling favorable winds are weak. Here, we show that in the tropical Angolan upwelling system (tAUS), the seasonal productivity maximum is due to the combined effect of coastal trapped waves (CTWs) and elevated tidal mixing on the shelf. During austral winter, the passage of an upwelling CTW displaces the nitracline upward by more than 50 m. Thereby, nitrate-rich waters spread onto the shelf, where elevated vertical mixing causes a nitrate flux into the surface mixed layer. Interannual variability of the productivity maximum is strongly correlated to the amplitude of the upwelling CTW as seen in sea level data. Given that CTWs are connected to equatorial forcing, a predictability of the strength of the productivity maximum is suggested.

Drivers and impact of the 2021 extreme warm event in the tropical Angolan upwelling system.

Benguela Niños are extreme warm events that typically occur during the main downwelling season (austral fall) in the tropical Angolan upwelling system when the biological productivity is low. However, the extreme warm event that occurred between April and August 2021 stands out due to its late timing. It occurred and peaked during the main upwelling season in austral winter with sea surface temperature anomalies exceeding 2 °C in the Angola-Benguela area in June 2021. This led to an unprecedented reduction of the net primary production off southern Angola. Both local atmospheric processes and remote influences (via downwelling coastal trapped wave propagations) have contributed to the onset of the extreme warm event in April and its intensification towards the peak phase in June. Moreover, the poleward advection of warm equatorial waters toward the Angola-Benguela area in May 2021 might have contributed to the warming, since the transport of the Angola Current, as estimated from observations, was notably elevated, amounting to 2.1 Sv.

Enhanced reporting of deaths among Aboriginal and Torres Strait Islander peoples using linked administrative health datasets.

BACKGROUND: Aboriginal and Torres Strait Islander peoples are under-reported in administrative health datasets in NSW, Australia. Correct reporting of Aboriginal and Torres Strait Islander peoples is essential to measure the effectiveness of policies and programmes aimed at reducing the health disadvantage experienced by Aboriginal and Torres Strait Islander peoples. This study investigates the potential of record linkage to enhance reporting of deaths among Aboriginal and Torres Strait Islander peoples in NSW, Australia. METHODS: Australian Bureau of Statistics death registration data for 2007 were linked with four population health datasets relating to hospitalisations, emergency department attendances and births. Reporting of deaths was enhanced from linked records using two methods, and effects on patterns of demographic characteristics and mortality indicators were examined. RESULTS: Reporting of deaths increased by 34.5% using an algorithm based on a weight of evidence of a person being Aboriginal or Torres Strait Islander, and by 56.6% using an approach based on 'at least one report' of a person being Aboriginal or Torres Strait Islander. The increase was relatively greater in older persons and those living in less geographically remote areas. Enhancement resulted in a reduction in the urban-remote differential in median age at death and increases in standardised mortality ratios particularly for chronic conditions. CONCLUSIONS: Record linkage creates a statistical construct that helps to correct under-reporting of deaths and potential bias in mortality statistics for Aboriginal and Torres Strait Islander peoples.

Diastereoselective Synthesis of N-Methylspiroindolines by Intramolecular Mizoroki-Heck Annulations.

Spiroindolines represent a privileged structure in medicinal chemistry, although stereocontrol around the spirocarbon can be a synthetic challenge. Here we present a palladium(0)-catalyzed intramolecular Mizoroki-Heck annulation reaction from (+)-Vince lactam-derived cyclopentenyl-tethered 2-bromo-N-methylanilines for the formation of N-methylspiroindolines. A series of 14 N-methylspiroindolines were synthesized in 59-81% yield with diastereoselectivity >98%, which was rationalized by density functional theory calculations and confirmed through X-ray crystallography. One spiroindoline was converted to an N- and C-terminal protected rigidified unnatural amino acid, which could be orthogonally deprotected.

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.

Imitation of ß-lactam binding enables broad-spectrum metallo-ß-lactamase inhibitors.

Carbapenems are vital antibiotics, but their efficacy is increasingly compromised by metallo-ß-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 ß-lactamase stable ß-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.

Surface cooling caused by rare but intense near-inertial wave induced mixing in the tropical Atlantic.

The direct response of the tropical mixed layer to near-inertial waves (NIWs) has only rarely been observed. Here, we present upper-ocean turbulence data that provide evidence for a strongly elevated vertical diffusive heat flux across the base of the mixed layer in the presence of a NIW, thereby cooling the mixed layer at a rate of 244 W m-2 over the 20 h of continuous measurements. We investigate the seasonal cycle of strong NIW events and find that despite their local intermittent nature, they occur preferentially during boreal summer, presumably associated with the passage of atmospheric African Easterly Waves. We illustrate the impact of these rare but intense NIW induced mixing events on the mixed layer heat balance, highlight their contribution to the seasonal evolution of sea surface temperature, and discuss their potential impact on biological productivity in the tropical North Atlantic.

Small kinase assay panels can provide a measure of selectivity.

In this Letter, a novel strategy for assessment of ligand promiscuity is described. By using a carefully selected small set of kinases together with multivariate statistical methods, a measure of selectivity can be estimated. This will facilitate an appropriate selection of compounds for further development in lead generation and optimization.

Pan-NS3 protease inhibitors of hepatitis C virus based on an R3-elongated pyrazinone scaffold.

Herein, we present the design and synthesis of 2(1H)-pyrazinone based HCV NS3 protease inhibitors and show that elongated R3 urea substituents were associated with increased inhibitory potencies over several NS3 protein variants. The inhibitors are believed to rely on ß-sheet mimicking hydrogen bonds which are similar over different genotypes and current drug resistant variants and correspond to the ß-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 1a, both wild-type (Ki = 30 nM) and R155K (Ki = 2 nM), and genotype 3a (Ki = 5 nM).