Identification of 10 differentially expressed and cuproptosis-related genes in immune infiltration and prognosis of thyroid carcinoma.

The association between the cuproptosis-related genes and the immune infiltration and their prognostic value in thyroid carcinoma is still unexplored. Bioinformatics analyses were performed with data obtained from the TCGA dataset. The aberrantly expressed genes were selected. KEGG and GO analyses were conducted to explore the enriched pathways of the up-regulated or down-regulated genes in thyroid carcinoma. Totally 1495 genes were differentially expressed (691 up-regulated, 804 down-regulated) in thyroid carcinoma (p<0.05). The 10 cuproptosis-related RNAs (DLD, LIAS, LIPT1, FDX1, DLAT, MTF1, PDHA1, CDKN2A, GLS and PDHB) were also demonstrated to be aberrantly expressed in thyroid carcinoma patients tissues. FDX1 expression was correlated with the overall survival in thyroid carcinoma patients (HR=0.4995, 95% CI: 0.2688-0.9285, p=0.0282). Further multivariate cox regression analysis revealed that DLD (HR=24.8869, 95% CI: 4.48772-138.01181, p=0.00024), and LIAS (HR=7.74092, 95% CI: 1.12194-53.40898, p=0.03783) were associated with the survival of thyroid carcinoma patients. The immune infiltration analysis demonstrated that significant correlation between the 10 cuproptosis-related genes and immune infiltration in thyroid carcinoma (p<0.01). We presented the expression profiles of dysregulated genes in thyroid carcinoma. The findings of our study highlighted the potential of cuproptosis-related genes as prognostic biomarkers for thyroid carcinoma.

Prognostic Stratification Based on a Novel Nomogram for Solitary Large Hepatocellular Carcinoma After Curative Resection.

Solitary large hepatocellular carcinoma (SLHCC) is a specific subtype of HCC with unique characteristics. It is of great interest to assess and stratify the prognosis of SLHCCs after curative resection. In this study, we tried to construct a prognostic nomogram for SLHCC following curative resection through a retrospective analysis of 202 SLHCC cases. Seven prognostic factors were identified and integrated to establish a novel prognostic nomogram, which included tumor size, microvascular invasion, tumor differentiation, Ki67 (%), α-fetoprotein (AFP), carbohydrate antigen 125 (CA125), and HBsAg status. The Harrell's concordance index (C-index) of the nomogram for overall survival (OS) in the training, validation, and whole sets was 0.752, 0.703, and 0.733, respectively. Furthermore, the area under the curve (AUC) of the receiver operating characteristic (ROC) curve of the nomogram for predicting 1-, 3-, and 5-year OS indicated that the nomogram had an optimal discrimination of the prognostic prediction for SLHCC. The total score of each patient was calculated based on the nomogram, and patients were divided into three subgroups: low-risk group (total score ≦ 107), medium-risk group (107 < total score ≤ 125), and high-risk group (total score > 125). The 1-, 3-, and 5-year OS rates of the low-risk, medium-risk, and high-risk groups in the whole set were 89.3 vs. 70.1 vs. 33.3%, 76.6 vs. 37.8 vs. 14.5%, and 69.8 vs. 25.1 vs. 12.5%, respectively (P < 0.001). Similar results were shown in terms of the recurrence-free survival (RFS) rate. By analyzing 101 cases of recurrent tumors, transarterial chemoembolization (TACE) plus radiofrequency ablation (RFA)/surgery was found to prolong patient survival when compared to TACE alone in the low-risk group, but not in the medium/high-risk group. In conclusion, our prognostic nomogram successfully stratifies the prognosis for SLHCC after curative resection, which deserves further study in future clinical practice.

MiR-142-5p Acts as a Significant Regulator Through Promoting Proliferation, Invasion, and Migration in Breast Cancer Modulated by Targeting SORBS1.

BACKGROUND: Numerous researches have demonstrated that miR-142-5p plays significant roles in several cancers, although the functional characteristic of miR-142-5p in breast cancer has not been determined. This study is designed to explore the biological significance of miR-142-5p in breast cancer clinical implication and mechanism of action. METHODS: The differential expression patterns of miR-142-5p and Sorbin and SH3 domain-containing protein 1 and correlations between them and clinical significances were analyzed based on data from database. The expression levels of miR-142-5p in breast cancer cells were detected using quantitative real-time polymerase chain reaction. Cell counting kit-8, transwell, and wound healing assays were used to explore the potential functions of miR-142-5p in breast cancer cells. In addition, bioinformatics prediction analysis and luciferase reporter assay were utilized to predict and identify the potential target gene of miR-142-5p. A rescue experiment was conducted by transfecting miR-142-5p inhibitors and si-Sorbin and SH3 domain-containing protein 1 into cells to explore miR-142-5p/Sorbin and SH3 domain-containing protein 1 pairs on breast cancer cells behaviors. RESULTS: The analysis results showed that miR-142-5p was highly expressed in patients with breast cancer, while Sorbin and SH3 domain-containing protein 1 presented a trend of low expression. The clinical significances analysis suggested that the overexpression of miR-142-5p is closely correlated with metastasis, while low expression of Sorbin and SH3 domain-containing protein 1 is correlated with clinicopathological characteristics and poor overall survival in patients with breast cancer. In vitro exploration, the expression of miR-142-5p was upregulated in breast cancer cells and inhibition of miR-142-5p expression significantly reduced the proliferation, invasion, and migration of breast cancer cells. Through rescue experiments, breast cancer cells proliferation, invasion, and migration reduction induced by silencing of miR-142-5p were reversed via knockdown Sorbin and SH3 domain-containing protein 1. CONCLUSION: Our findings insinuate that miR-142-5p functions as a positive regulator of promoting breast cancer cells biological behaviors and clinical metastasis, possibly regulated by targeting Sorbin and SH3 domain-containing protein 1, thus providing valuable information in the development of preventive or even therapeutic strategies for utilizing miR-142-5p as a promising target.

Ecosystem health monitoring in the Shanghai-Hangzhou Bay Metropolitan Area: A hidden Markov modeling approach.

Ecosystem health assessment is an important method for obtaining information on ecosystem conditions, and it plays a vital role in preserving and enhancing ecosystem health status. In addition, it provides useful information and knowledge for urban agglomeration development decision makers. However, ecological phenomena often vary considerably from one observation to the next, which makes it difficult to distinguish different status of the ecosystem health. In this study, hidden Markov model (HMM) was employed to simulate the internal-external correlations of ecosystem status through establishing the relationships between internal ecological health level and combination state of external observation. Based on the statistics and land use data in 2001, 2007 and 2013, the Vigor-Organization-Resilience (VOR) framework was employed to identify the ecosystem health in Shanghai-Hangzhou Bay Metropolitan (SHBM), in which the ecosystem health state was considered as a hidden state that could be estimated according to the conditions of vigor, organization and resilience. In addition, two parameter learning cases including mathematical statistics and extensible sequence method were employed to solve the iterative convergence problem of parameters in short-time series of ecosystem health simulation. Results show that HMM not only provides a comparable descriptive ability to that of the VOR model, but also can monitor ecosystem health at the optimal grid scale in SHBM. The combination of HMM and VOR greatly expands the spatiotemporal characteristics and provides a new research approach for the study of ecosystem health assessment of urban agglomerations.

An integrated bioinformatics analysis of potential therapeutic targets among matrix metalloproteinases in breast cancer.

Breast cancer (BC) is one of the most aggressive malignancies worldwide among females. Matrix metalloproteinases (MMPs), as the most abundant class of non-serine proteases present in invasive and metastatic tumors, can regulate a variety of alterations in the microenvironment during tumor progression. However, the differential expression of MMPs and its prognostic values in BC is yet to be elucidated. In this research, using the ONCOMINE dataset, The Cancer Genome Atlas, Breast Cancer Gene-Expression Miner v4.1 (Bc-GenExMiner), Kaplan-Meier Plotter and cBioPortal, the transcriptional MMPs and survival outcome data of patients with BC was compared. It was indicated that mRNA levels of MMP1/3/9/10/11/12/13 were increased compared with non-tumor tissues, whereas mRNA expression of MMP2/16/19/23B/28 was lower in BC tissues. Kaplan-Meier plots showed that high mRNA levels of MMP2/10/16/19/20/23B/27 in patients with BC were associated with better recurrence-free survival. In contrast, high MMP1/8/9/11/12 conferred worse RFS rate. Meanwhile, high transcription levels of MMP1/3/11/12/13 predicted shorter distant metastasis-free survival, while high levels of MMP1/12 demonstrated worse overall survival in patients with BC. From Bc-GenExMiner, it was indicated that high expression of MMP16/20 was correlated with better prognosis, while MMP1/9/11/12/13/14/15 exerted a negative effect on patient prognosis. The integrative bioinformatics analysis performed in the present study suggests that MMP1/9/12/16, compared with other MMPs, are potentially appropriate targets for targeted therapy in patients with BC.

Correction to "Discovery of Irreversible Inhibitors Targeting Histone Methyltransferase, SMYD3".

[This corrects the article DOI: 10.1021/acsmedchemlett.9b00170.].

Discovery of Irreversible Inhibitors Targeting Histone Methyltransferase, SMYD3.

SMYD3 is a histone methyltransferase that regulates gene transcription, and its overexpression is associated with multiple human cancers. A novel class of tetrahydroacridine compounds which inhibit SMYD3 through a covalent mechanism of action is identified. Optimization of these irreversible inhibitors resulted in the discovery of 4-chloroquinolines, a new class of covalent warheads. Tool compound 29 exhibits high potency by inhibiting SMYD3's enzymatic activity and showing antiproliferative activity against HepG2 in 3D cell culture. Our findings suggest that covalent inhibition of SMYD3 may have an impact on SMYD3 biology by affecting expression levels, and this warrants further exploration.

miR-501 acts as an independent prognostic factor that promotes the epithelial-mesenchymal transition through targeting JDP2 in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC), the second common cancer, was a kind of primary liver cancer with high incidence. miR-501, identified as a novel regulator, was acted as a potential biomarker in several diseases. JDP2, acted as a repressor of AP-1 complex, was a member of the basic leucine zipper (bZIP) transcription factor family. RT-qPCR was applied to evaluate miR-501 and JDP2 expression level and we found that miR-501 was upregulated in HCC tissues and cells. miR-501 ectopic expression promoted HCC cell invasion and epithelial-mesenchymal transition (EMT), while low expression present the opposite results. JDP2 was downregulated in HCC tissues and cells, and overexpressed JDP2 facilitated HCC cell invasion and EMT. Furthermore, luciferase reporter assay indicated that JDP2 was a target of miR-501 and altered miR-501 expression the JPD2 mRNA may changed. The expression of miR-501 and JDP2 had negative connection in HCC tissues. In addition, Kaplan-Meier method revealed that miR-501 upregulation or JDP2 downregulation predicted poor prognosis in HCC patients. miR-501 promoted cell invasion and EMT by regulated JDP2 in hepatocellular carcinoma. The newly identified miR-501/JDP2 axis provides novel insight into the pathogenesis of hepatocellular carcinoma.

Cholesterol goal achievement and lipid-lowering therapy in patients with stable or acute coronary heart disease in Singapore: results from the Dyslipidemia International Study II.

INTRODUCTION: Dyslipidaemia is a major risk factor for coronary heart disease (CHD). There is a lack of data on the extent of lipid abnormalities and lipid-lowering therapy (LLT) in Singapore. METHODS: The Dyslipidemia International Study (DYSIS) II was a multinational observational study of patients with stable CHD and hospitalised patients with an acute coronary syndrome (ACS). A full lipid profile and use of LLT were documented at baseline, and for the ACS cohort, at four months post-hospitalisation. RESULTS: 325 patients were recruited from four sites in Singapore; 199 had stable CHD and 126 were hospitalised with an ACS. At baseline, 96.5% of the CHD cohort and 66.4% of the ACS cohort were being treated with LLT. In both cohorts, low-density lipoprotein cholesterol (LDL-C) levels were lower for the treated than the non-treated patients; accordingly, a higher proportion of patients met the LDL-C goal of < 70 mg/dL (CHD: 28.1% vs. 0%, p = 0.10; ACS: 20.2% vs. 0%, p < 0.01). By the four-month follow-up, a higher proportion of the ACS patients that were originally not treated with LLT had met the LDL-C goal (from 0% to 54.5%), correlating with the increased use of medication. However, there was negligible improvement in the patients who were treated prior to the ACS. CONCLUSION: Dyslipidaemia is a significant concern in Singapore, with few patients with stable or acute CHD meeting the recommended European Society of Cardiology/European Atherosclerosis Society goal. LLT was widely used but not optimised, indicating considerable scope for improved management of these very-high-risk patients.

Evaluating the Contribution of Transition-State Destabilization to Changes in the Residence Time of Triazole-Based InhA Inhibitors.

A critical goal of lead compound selection and optimization is to maximize target engagement while minimizing off-target binding. Since target engagement is a function of both the thermodynamics and kinetics of drug-target interactions, it follows that the structures of both the ground states and transition states on the binding reaction coordinate are needed to rationally modulate the lifetime of the drug-target complex. Previously, we predicted the structure of the rate-limiting transition state that controlled the time-dependent inhibition of the enoyl-ACP reductase InhA. This led to the discovery of a triazole-containing diphenyl ether with an increased residence time on InhA due to transition-state destabilization rather than ground-state stabilization. In the present work, we evaluate the inhibition of InhA by 14 triazole-based diphenyl ethers and use a combination of enzyme kinetics and X-ray crystallography to generate a structure-kinetic relationship for time-dependent binding. We show that the triazole motif slows the rate of formation for the final drug-target complex by up to 3 orders of magnitude. In addition, we identify a novel inhibitor with a residence time on InhA of 220 min, which is 3.5-fold longer than that of the INH-NAD adduct formed by the tuberculosis drug, isoniazid. This study provides a clear example in which the lifetime of the drug-target complex is controlled by interactions in the transition state for inhibitor binding rather than the ground state of the enzyme-inhibitor complex, and demonstrates the important role that on-rates can play in drug-target residence time.

Rationalizing the Binding Kinetics for the Inhibition of the Burkholderia pseudomallei FabI1 Enoyl-ACP Reductase.

There is growing awareness of the link between drug-target residence time and in vivo drug activity, and there are increasing efforts to determine the molecular factors that control the lifetime of a drug-target complex. Rational alterations in the drug-target residence time require knowledge of both the ground and transition states on the inhibition reaction coordinate, and we have determined the structure-kinetic relationship for 22 ethyl- or hexyl-substituted diphenyl ethers that are slow-binding inhibitors of bpFabI1, the enoyl-ACP reductase FabI1 from Burkholderia pseudomallei. Analysis of enzyme inhibition using a two-dimensional kinetic map demonstrates that the ethyl and hexyl diphenyl ethers fall into two distinct clusters. Modifications to the ethyl diphenyl ether B ring result in changes to both on and off rates, where residence times of up to ∼700 min (∼11 h) are achieved by either ground state stabilization (PT444) or transition state destabilization (slower on rate) (PT404). By contrast, modifications to the hexyl diphenyl ether B ring result in residence times of 300 min (∼5 h) through changes in only ground state stabilization (PT119). Structural analysis of nine enzyme:inhibitor complexes reveals that the variation in structure-kinetic relationships can be rationalized by structural rearrangements of bpFabI1 and subtle changes to the orientation of the inhibitor in the binding pocket. Finally, we demonstrate that three compounds with residence times on bpFabI1 from 118 min (∼2 h) to 670 min (∼11 h) have in vivo efficacy in an acute B. pseudomallei murine infection model using the virulent B. pseudomallei strain Bp400.

Rational Modulation of the Induced-Fit Conformational Change for Slow-Onset Inhibition in Mycobacterium tuberculosis InhA.

Slow-onset enzyme inhibitors are the subject of considerable interest as an approach to increasing the potency of pharmaceutical compounds by extending the residence time of the inhibitor on the target (the lifetime of the drug-receptor complex). However, rational modulation of residence time presents significant challenges because it requires additional mechanistic insight, such as the nature of the transition state for postbinding isomerization. Our previous work, based on X-ray crystallography, enzyme kinetics, and molecular dynamics simulation, suggested that the slow step in inhibition of the Mycobacterium tuberculosis enoyl-ACP reductase InhA involves a change in the conformation of the substrate binding loop from an open state in the initial enzyme-inhibitor complex to a closed state in the final enzyme-inhibitor complex. Here, we use multidimensional free energy landscapes for loop isomerization to obtain a computational model for the transition state. The results suggest that slow-onset inhibitors crowd key side chains on helices that slide past each other during isomerization, resulting in a steric clash. The landscapes become significantly flatter when residues involved in the steric clash are replaced with alanine. Importantly, this lower barrier can be increased by rational inhibitor redesign to restore the steric clash. Crystallographic studies and enzyme kinetics confirm the predicted effects on loop structure and flexibility, as well as inhibitor residence time. These loss and regain of function studies validate our mechanistic hypothesis for interactions controlling substrate binding loop isomerization, providing a platform for the future design of inhibitors with longer residence times and better in vivo potency. Similar opportunities for slow-onset inhibition via the same mechanism are identified in other pathogens.

A [(32)P]NAD(+)-based method to identify and quantitate long residence time enoyl-acyl carrier protein reductase inhibitors.

The classical methods for quantifying drug-target residence time (tR) use loss or regain of enzyme activity in progress curve kinetic assays. However, such methods become imprecise at very long residence times, mitigating the use of alternative strategies. Using the NAD(P)H-dependent FabI enoyl-acyl carrier protein (enoyl-ACP) reductase as a model system, we developed a Penefsky column-based method for direct measurement of tR, where the off-rate of the drug was determined with radiolabeled [adenylate-(32)P]NAD(P(+)) cofactor. In total, 23 FabI inhibitors were analyzed, and a mathematical model was used to estimate limits to the tR values of each inhibitor based on percentage drug-target complex recovery following gel filtration. In general, this method showed good agreement with the classical steady-state kinetic methods for compounds with tR values of 10 to 100 min. In addition, we were able to identify seven long tR inhibitors (100-1500 min) and to accurately determine their tR values. The method was then used to measure tR as a function of temperature, an analysis not previously possible using the standard kinetic approach due to decreased NAD(P)H stability at elevated temperatures. In general, a 4-fold difference in tR was observed when the temperature was increased from 25 to 37 °C.

An ordered water channel in Staphylococcus aureus FabI: unraveling the mechanism of substrate recognition and reduction.

One third of all drugs in clinical use owe their pharmacological activity to the functional inhibition of enzymes, highlighting the importance of enzymatic targets for drug development. Because of the close relationship between inhibition and catalysis, understanding the recognition and turnover of enzymatic substrates is essential for rational drug design. Although the Staphylococcus aureus enoyl-acyl carrier protein reductase (saFabI) involved in bacterial fatty acid biosynthesis constitutes a very promising target for the development of novel, urgently needed anti-staphylococcal agents, the substrate binding mode and catalytic mechanism remained unclear for this enzyme. Using a combined crystallographic, kinetic, and computational approach, we have explored the chemical properties of the saFabI binding cavity, obtaining a consistent mechanistic model for substrate binding and turnover. We identified a water-molecule network linking the active site with a water basin inside the homo-tetrameric protein, which seems to be crucial for the closure of the flexible substrate binding loop as well as for an effective hydride and proton transfer during catalysis. On the basis of our results, we also derive a new model for the FabI-ACP complex that reveals how the ACP-bound acyl-substrate is injected into the FabI binding crevice. These findings support the future development of novel FabI inhibitors that target the FabI-ACP interface leading to the disruption of the interaction between these two proteins.

Direct inhibitors of InhA are active against Mycobacterium tuberculosis.

New chemotherapeutic agents are urgently required to combat the global spread of multidrug-resistant tuberculosis (MDR-TB). The mycobacterial enoyl reductase InhA is one of the few clinically validated targets in tuberculosis drug discovery. We report the identification of a new class of direct InhA inhibitors, the 4-hydroxy-2-pyridones, using phenotypic high-throughput whole-cell screening. This class of orally active compounds showed potent bactericidal activity against common isoniazid-resistant TB clinical isolates. Biophysical studies revealed that 4-hydroxy-2-pyridones bound specifically to InhA in an NADH (reduced form of nicotinamide adenine dinucleotide)-dependent manner and blocked the enoyl substrate-binding pocket. The lead compound NITD-916 directly blocked InhA in a dose-dependent manner and showed in vivo efficacy in acute and established mouse models of Mycobacterium tuberculosis infection. Collectively, our structural and biochemical data open up new avenues for rational structure-guided optimization of the 4-hydroxy-2-pyridone class of compounds for the treatment of MDR-TB.

A virtual screen discovers novel, fragment-sized inhibitors of Mycobacterium tuberculosis InhA.

Isoniazid (INH) is usually administered to treat latent Mycobacterium tuberculosis (Mtb) infections and is used in combination therapy to treat active tuberculosis (TB). Unfortunately, resistance to this drug is hampering its clinical effectiveness. INH is a prodrug that must be activated by Mtb catalase-peroxidase (KatG) before it can inhibit InhA (Mtb enoyl-acyl-carrier-protein reductase). Isoniazid-resistant cases of TB found in clinical settings usually involve mutations in or deletion of katG, which abrogate INH activation. Compounds that inhibit InhA without requiring prior activation by KatG would not be affected by this resistance mechanism and hence would display continued potency against these drug-resistant isolates of Mtb. Virtual screening experiments versus InhA in the GO Fight Against Malaria (GO FAM) project were designed to discover new scaffolds that display base-stacking interactions with the NAD cofactor. GO FAM experiments included targets from other pathogens, including Mtb, when they had structural similarity to a malaria target. Eight of the 16 soluble compounds identified by docking against InhA plus visual inspection were modest inhibitors and did not require prior activation by KatG. The best two inhibitors discovered are both fragment-sized compounds and displayed Ki values of 54 and 59 µM, respectively. Importantly, the novel inhibitors discovered have low structural similarity to known InhA inhibitors and thus help expand the number of chemotypes on which future medicinal chemistry efforts can be focused. These new fragment hits could eventually help advance the fight against INH-resistant Mtb strains, which pose a significant global health threat.

Rational design of broad spectrum antibacterial activity based on a clinically relevant enoyl-acyl carrier protein (ACP) reductase inhibitor.

Determining the molecular basis for target selectivity is of particular importance in drug discovery. The ideal antibiotic should be active against a broad spectrum of pathogenic organisms with a minimal effect on human targets. CG400549, a Staphylococcus-specific 2-pyridone compound that inhibits the enoyl-acyl carrier protein reductase (FabI), has recently been shown to possess human efficacy for the treatment of methicillin-resistant Staphylococcus aureus infections, which constitute a serious threat to human health. In this study, we solved the structures of three different FabI homologues in complex with several pyridone inhibitors, including CG400549. Based on these structures, we rationalize the 65-fold reduced affinity of CG400549 toward Escherichia coli versus S. aureus FabI and implement concepts to improve the spectrum of antibacterial activity. The identification of different conformational states along the reaction coordinate of the enzymatic hydride transfer provides an elegant visual depiction of the relationship between catalysis and inhibition, which facilitates rational inhibitor design. Ultimately, we developed the novel 4-pyridone-based FabI inhibitor PT166 that retained favorable pharmacokinetics and efficacy in a mouse model of S. aureus infection with extended activity against Gram-negative and mycobacterial organisms.

A structural and energetic model for the slow-onset inhibition of the Mycobacterium tuberculosis enoyl-ACP reductase InhA.

Slow-onset enzyme inhibitors are of great interest for drug discovery programs since the slow dissociation of the inhibitor from the drug-target complex results in sustained target occupancy leading to improved pharmacodynamics. However, the structural basis for slow-onset inhibition is often not fully understood, hindering the development of structure-kinetic relationships and the rational optimization of drug-target residence time. Previously we demonstrated that slow-onset inhibition of the Mycobacterium tuberculosis enoyl-ACP reductase InhA correlated with motions of a substrate-binding loop (SBL) near the active site. In the present work, X-ray crystallography and molecular dynamics simulations have been used to map the structural and energetic changes of the SBL that occur upon enzyme inhibition. Helix-6 within the SBL adopts an open conformation when the inhibitor structure or binding kinetics is substrate-like. In contrast, slow-onset inhibition results in large-scale local refolding in which helix-6 adopts a closed conformation not normally populated during substrate turnover. The open and closed conformations of helix-6 are hypothesized to represent the EI and EI* states on the two-step induced-fit reaction coordinate for enzyme inhibition. These two states were used as the end points for nudged elastic band molecular dynamics simulations resulting in two-dimensional potential energy profiles that reveal the barrier between EI and EI*, thus rationalizing the binding kinetics observed with different inhibitors. Our findings indicate that the structural basis for slow-onset kinetics can be understood once the structures of both EI and EI* have been identified, thus providing a starting point for the rational control of enzyme-inhibitor binding kinetics.

Substituted diphenyl ethers as a novel chemotherapeutic platform against Burkholderia pseudomallei.

Identification of a novel class of anti-Burkholderia compounds is key in addressing antimicrobial resistance to current therapies as well as naturally occurring resistance. The FabI enoyl-ACP reductase in Burkholderia is an underexploited target that presents an opportunity for development of a new class of inhibitors. A library of substituted diphenyl ethers was used to identify FabI1-specific inhibitors for assessment in Burkholderia pseudomallei ex vivo and murine efficacy models. Active FabI1 inhibitors were identified in a two-stage format consisting of percent inhibition screening and MIC determination by the broth microdilution method. Each compound was evaluated against the B. pseudomallei 1026b (efflux-proficient) and Bp400 (efflux-compromised) strains. In vitro screening identified candidate substituted diphenyl ethers that exhibited MICs of less than 1 µg/ml, and enzyme kinetic assays were used to assess potency and specificity against the FabI1 enzyme. These compounds demonstrated activity in a Burkholderia ex vivo efficacy model, and two demonstrated efficacy in an acute B. pseudomallei mouse infection model. This work establishes substituted diphenyl ethers as a suitable platform for development of novel anti-Burkholderia compounds that can be used for treatment of melioidosis.

Rational optimization of drug-target residence time: insights from inhibitor binding to the Staphylococcus aureus FabI enzyme-product complex.

Drug-target kinetics has recently emerged as an especially important facet of the drug discovery process. In particular, prolonged drug-target residence times may confer enhanced efficacy and selectivity in the open in vivo system. However, the lack of accurate kinetic and structural data for a series of congeneric compounds hinders the rational design of inhibitors with decreased off-rates. Therefore, we chose the Staphylococcus aureus enoyl-ACP reductase (saFabI)--an important target for the development of new anti-staphylococcal drugs--as a model system to rationalize and optimize the drug-target residence time on a structural basis. Using our new, efficient, and widely applicable mechanistically informed kinetic approach, we obtained a full characterization of saFabI inhibition by a series of 20 diphenyl ethers complemented by a collection of 9 saFabI-inhibitor crystal structures. We identified a strong correlation between the affinities of the investigated saFabI diphenyl ether inhibitors and their corresponding residence times, which can be rationalized on a structural basis. Because of its favorable interactions with the enzyme, the residence time of our most potent compound exceeds 10 h. In addition, we found that affinity and residence time in this system can be significantly enhanced by modifications predictable by a careful consideration of catalysis. Our study provides a blueprint for investigating and prolonging drug-target kinetics and may aid in the rational design of long-residence-time inhibitors targeting the essential saFabI enzyme.