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1.
Pharmaceuticals (Basel) ; 16(1)2023 Jan 04.
Article in English | MEDLINE | ID: mdl-36678572

ABSTRACT

Hyperactive mutation V617F in the JAK2 regulatory pseudokinase domain (JH2) is prevalent in patients with myeloproliferative neoplasms. Here, we identified novel small molecules that target JH2 of JAK2 V617F and characterized binding via biochemical and structural approaches. Screening of 107,600 small molecules resulted in identification of 55 binders to the ATP-binding pocket of recombinant JAK2 JH2 V617F protein at a low hit rate of 0.05%, which indicates unique structural characteristics of the JAK2 JH2 ATP-binding pocket. Selected hits and structural analogs were further assessed for binding to JH2 and JH1 (kinase) domains of JAK family members (JAK1-3, TYK2) and for effects on MPN model cell viability. Crystal structures were determined with JAK2 JH2 wild-type and V617F. The JH2-selective binders were identified in diaminotriazole, diaminotriazine, and phenylpyrazolo-pyrimidone chemical entities, but they showed low-affinity, and no inhibition of MPN cells was detected, while compounds binding to both JAK2 JH1 and JH2 domains inhibited MPN cell viability. X-ray crystal structures of protein-ligand complexes indicated generally similar binding modes between the ligands and V617F or wild-type JAK2. Ligands of JAK2 JH2 V617F are applicable as probes in JAK-STAT research, and SAR optimization combined with structural insights may yield higher-affinity inhibitors with biological activity.

2.
Nat Cancer ; 3(2): 156-172, 2022 02.
Article in English | MEDLINE | ID: mdl-35228749

ABSTRACT

The folate metabolism enzyme MTHFD2 (methylenetetrahydrofolate dehydrogenase/cyclohydrolase) is consistently overexpressed in cancer but its roles are not fully characterized, and current candidate inhibitors have limited potency for clinical development. In the present study, we demonstrate a role for MTHFD2 in DNA replication and genomic stability in cancer cells, and perform a drug screen to identify potent and selective nanomolar MTHFD2 inhibitors; protein cocrystal structures demonstrated binding to the active site of MTHFD2 and target engagement. MTHFD2 inhibitors reduced replication fork speed and induced replication stress followed by S-phase arrest and apoptosis of acute myeloid leukemia cells in vitro and in vivo, with a therapeutic window spanning four orders of magnitude compared with nontumorigenic cells. Mechanistically, MTHFD2 inhibitors prevented thymidine production leading to misincorporation of uracil into DNA and replication stress. Overall, these results demonstrate a functional link between MTHFD2-dependent cancer metabolism and replication stress that can be exploited therapeutically with this new class of inhibitors.


Subject(s)
Aminohydrolases , Leukemia, Myeloid, Acute , Aminohydrolases/genetics , Humans , Hydrolases , Leukemia, Myeloid, Acute/drug therapy , Methylenetetrahydrofolate Dehydrogenase (NADP)/genetics , Multifunctional Enzymes/genetics , Thymidine
3.
J Am Chem Soc ; 144(7): 2905-2920, 2022 02 23.
Article in English | MEDLINE | ID: mdl-35142215

ABSTRACT

Drugs targeting SARS-CoV-2 could have saved millions of lives during the COVID-19 pandemic, and it is now crucial to develop inhibitors of coronavirus replication in preparation for future outbreaks. We explored two virtual screening strategies to find inhibitors of the SARS-CoV-2 main protease in ultralarge chemical libraries. First, structure-based docking was used to screen a diverse library of 235 million virtual compounds against the active site. One hundred top-ranked compounds were tested in binding and enzymatic assays. Second, a fragment discovered by crystallographic screening was optimized guided by docking of millions of elaborated molecules and experimental testing of 93 compounds. Three inhibitors were identified in the first library screen, and five of the selected fragment elaborations showed inhibitory effects. Crystal structures of target-inhibitor complexes confirmed docking predictions and guided hit-to-lead optimization, resulting in a noncovalent main protease inhibitor with nanomolar affinity, a promising in vitro pharmacokinetic profile, and broad-spectrum antiviral effect in infected cells.


Subject(s)
Antiviral Agents/pharmacology , Coronavirus 3C Proteases/metabolism , Cysteine Proteinase Inhibitors/pharmacology , SARS-CoV-2/drug effects , Small Molecule Libraries/pharmacology , Animals , Antiviral Agents/metabolism , Antiviral Agents/pharmacokinetics , Catalytic Domain , Chlorocebus aethiops , Coronavirus 3C Proteases/chemistry , Cysteine Proteinase Inhibitors/metabolism , Cysteine Proteinase Inhibitors/pharmacokinetics , Drug Evaluation, Preclinical , Humans , Microbial Sensitivity Tests , Microsomes, Liver/metabolism , Molecular Docking Simulation , Protein Binding , SARS-CoV-2/enzymology , Small Molecule Libraries/metabolism , Small Molecule Libraries/pharmacokinetics , Vero Cells
4.
Prostaglandins Other Lipid Mediat ; 107: 26-34, 2013 Dec.
Article in English | MEDLINE | ID: mdl-24045148

ABSTRACT

Microsomal prostaglandin E synthase-1 (mPGES-1) inhibition has been suggested as an alternative to cyclooxygenase (COX) inhibition in the treatment of pain and inflammation. We characterized a selective inhibitor of mPGES-1 activity (compound III) and studied its impact on the prostanoid profile in various models of inflammation. Compound III is a benzoimidazole, which has a submicromolar IC50 in both human and rat recombinant mPGES-1. In cellular assays, it reduced PGE2 production in A549 cells, mouse macrophages and blood, causing a shunt to the prostacyclin pathway in the former two systems. Lastly, we assayed compound III in the air pouch model to verify its impact on the prostanoid profile and compare it to the profile obtained in mPGES-1 k.o. mice. As opposed to mPGES-1 genetic deletion, which attenuated PGE2 production and caused a shunt to the thromboxane pathway, mPGES-1 inhibition with compound III reduced PGE2 production and tended to decrease the levels of other prostanoids.


Subject(s)
Benzimidazoles/pharmacology , Enzyme Inhibitors/pharmacology , Intramolecular Oxidoreductases/antagonists & inhibitors , Isonipecotic Acids/pharmacology , Animals , Cell Line, Tumor , Dinoprostone/metabolism , Drug Evaluation, Preclinical , Gene Knockout Techniques , Humans , Inhibitory Concentration 50 , Intramolecular Oxidoreductases/genetics , Intramolecular Oxidoreductases/metabolism , Lipopolysaccharides/pharmacology , Macrophages, Peritoneal/enzymology , Macrophages, Peritoneal/immunology , Mice , Mice, Inbred DBA , Mice, Knockout , Prostaglandin H2/metabolism , Prostaglandin-E Synthases , Rats , Thromboxane B2/metabolism
5.
Assay Drug Dev Technol ; 9(5): 487-95, 2011 Oct.
Article in English | MEDLINE | ID: mdl-21561373

ABSTRACT

Microsomal prostaglandin E(2) synthase-1 (MPGES1) catalyzes the formation of prostaglandin E(2) from the endoperoxide prostaglandin H(2). MPGES1 expression is induced in inflammatory diseases, and this enzyme is regarded as a potential drug target. To aid in the drug discovery effort, a simple method for determination of inhibition mechanism and potency toward both prostaglandin H(2) and glutathione (GSH) has been developed. Using an assay with thiobarbituric acid-based detection, the inhibitory effects of six MPGES1 inhibitors were evaluated. The IC(50) values obtained at three substrate (S) concentrations ([S]K(M)) were used to estimate inhibition modality and inhibition constant values. This facilitated strategy is a useful and general screening method to evaluate the inhibitory effects of new drug compounds.


Subject(s)
Drug Discovery/methods , Drug Evaluation, Preclinical/methods , Drug Interactions , Enzyme Inhibitors/metabolism , Intramolecular Oxidoreductases/antagonists & inhibitors , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemistry , Fluorescence , Glutathione/analysis , Humans , Indoles/analysis , Indoles/pharmacokinetics , Indoles/pharmacology , Inhibitory Concentration 50 , Intramolecular Oxidoreductases/analysis , Intramolecular Oxidoreductases/physiology , Malondialdehyde/metabolism , Models, Theoretical , Molecular Targeted Therapy , Pharmacokinetics , Prostaglandin H2/antagonists & inhibitors , Prostaglandin H2/metabolism , Prostaglandin-E Synthases , Thiobarbiturates/metabolism
6.
J Mol Microbiol Biotechnol ; 9(2): 101-9, 2005.
Article in English | MEDLINE | ID: mdl-16319499

ABSTRACT

Antisense agents that inhibit genes at the mRNA level are attractive tools for genome-wide studies and drug target validation. The approach may be particularly well suited to studies of bacteria that are difficult to manipulate with standard genetic tools. Antisense peptide nucleic acids (PNA) with attached carrier peptides can inhibit gene expression in Escherichia coli and Staphylococcus aureus. Here we asked whether peptide-PNAs could mediate antisense effects in Mycobacterium smegmatis. We first targeted the gfp reporter gene and observed dose- and sequence-dependent inhibition at low micromolar concentrations. Sequence alterations within both the PNA and target mRNA sequences eliminated inhibition, strongly supporting an antisense mechanism of inhibition. Also, antisense PNAs with various attached peptides showed improved anti-gfp effects. Two peptide-PNAs targeted to the essential gene inhA were growth inhibitory and caused cell morphology changes that resemble that of InhA-depleted cells. Therefore, antisense peptide-PNAs can efficiently and specifically inhibit both reporter and endogenous essential genes in mycobacteria.


Subject(s)
Anti-Bacterial Agents/pharmacology , Antisense Elements (Genetics)/pharmacology , Gene Expression/drug effects , Mycobacterium smegmatis/drug effects , Peptide Nucleic Acids/pharmacology , Anti-Bacterial Agents/chemistry , Antisense Elements (Genetics)/genetics , Bacterial Proteins/drug effects , Down-Regulation/genetics , Green Fluorescent Proteins/analysis , Green Fluorescent Proteins/genetics , Mycobacterium smegmatis/genetics , Mycobacterium smegmatis/growth & development , Oxidoreductases/drug effects , Peptide Nucleic Acids/genetics , RNA, Messenger/analysis
7.
J Mol Biol ; 351(4): 776-83, 2005 Aug 26.
Article in English | MEDLINE | ID: mdl-16045927

ABSTRACT

Oligopyrimidine*oligopurine sequences with potential to form intramolecular triple helix structures (H-DNA) have been found mainly in high eukaryote genomes. However, the natural occurrence and function of H-DNA remains elusive largely because we lack appropriate reagents to demonstrate the formation of these structures in cells. We examined whether a triple-helix specific stabilizing compound, benzoquinoquinoxaline (BQQ), and its 1,10-phenanthroline derivative can be efficiently utilized to study the formation and stabilization of an intramolecular triple-helical DNA structure in growing Escherichia coli cells and in vitro. Cell uptake of BQQ was confirmed by fluorescence microscopy. A plasmid carrying an H-DNA forming sequence upstream of a reporter gene was used to assess the effects of H-DNA formation and stabilization in growing cells. The presence of the H-DNA forming sequence dramatically repressed beta-lactamase expression, and sub-growth-inhibitory doses of BQQ caused a further 40% reduction. Most importantly, repression was dependent on the triple-helix forming sequence and correlated with the addition of BQQ. As the abundance of the H-DNA forming plasmid was not affected by the addition of BQQ, the dose-dependent reduction at the protein level observed here is likely caused by repression of transcription. Finally, the triple-helix specific interaction of BQQ with the target DNA sequence was demonstrated using a triple-helix directed cleavage assay by BQQ-1,10-phenanthroline conjugate in vitro.


Subject(s)
DNA/chemistry , DNA/drug effects , Quinoxalines/pharmacology , Base Sequence , DNA/genetics , DNA, Bacterial/chemistry , DNA, Bacterial/drug effects , DNA, Bacterial/genetics , Drug Stability , Escherichia coli/drug effects , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression/drug effects , Nucleic Acid Conformation , Plasmids/genetics , Plasmids/metabolism , Quinoxalines/metabolism , Transcription, Genetic/drug effects
8.
J Antimicrob Chemother ; 56(1): 97-103, 2005 Jul.
Article in English | MEDLINE | ID: mdl-15914490

ABSTRACT

BACKGROUND: The few available distinct classes of antimicrobials limits the scope for single and combination drug treatment of resistant infections. OBJECTIVE: To evaluate antimicrobial effectiveness from combinations of protein-specific drugs and mRNA-specific antisense inhibitors. METHODS: Interactions between conventional antimicrobial drugs and mRNA-specific translation inhibiting antisense peptide nucleic acids were assessed in Escherichia coli and Staphylococcus aureus cultures using pairwise combinations in a chequerboard arrangement. Fractional inhibitory concentration indices (FICIs) were calculated and grouped according to the functional relationship between the inhibitor targets. Antisense specificity controls included different antisense sequences targeting the same mRNA, as well as biochemical quantification of active protein expressed from the essential fabI gene and from the lacZ reporter gene after single and combined inhibitor treatment. RESULTS: FICIs were higher for inhibitor combinations with unrelated targets than for combinations with functionally related targets. Inhibitor combinations with shared genetic targets displayed the lowest FICIs, with several qualifying for the conservative definition of antimicrobial synergy (FICI < or = 0.5). Furthermore, low FICIs arise as the hyperbolic dose-response curves for each separate inhibitor are maintained in combination. CONCLUSION: Interactions between mRNA- and protein-level inhibitors with the same genetic target can be synergistic and may provide a strategy to improve antimicrobial efficacy, facilitate drug mechanism of action studies and aid the search for new antimicrobials.


Subject(s)
Anti-Bacterial Agents/pharmacology , Antisense Elements (Genetics)/pharmacology , Peptide Nucleic Acids/pharmacology , RNA, Messenger/antagonists & inhibitors , Dose-Response Relationship, Drug , Drug Synergism , Microbial Sensitivity Tests
9.
Mol Ther ; 10(4): 652-9, 2004 Oct.
Article in English | MEDLINE | ID: mdl-15451449

ABSTRACT

Gene function studies in bacteria lag behind progress in genome sequencing, in part because current reverse genetics technology based on genome disruption does not allow subtle control of gene expression for all genes in a range of species. Essential genes and clustered regions are particularly problematic. Antisense technology offers an attractive alternative for microbial genomics. Unfortunately, bacteria lack RNAi mechanisms and conventional oligonucleotides are not taken up efficiently. However, in Escherichia coli, efficient and gene-specific antisense knock down is possible using antisense peptide nucleic acids (PNAs) attached to carrier peptides (KFFKFFKFFK). Carrier peptides can enter a range of microbial species, and in this study we asked whether peptide-PNAs could mediate antisense effects in Staphylococcus aureus. Using low micromolar concentrations we observed dose- and sequence-dependent inhibition of the reporter gene gfp and endogenous gene phoB. Also, antisense peptide-PNAs targeted to the essential genes fmhB, gyrA, and hmrB were growth inhibitory. Control peptide-PNAs were much less effective, and sequence alterations within the PNA and target mRNA sequences reduced or eliminated inhibition. Further development is needed to raise the antibacterial potential of PNAs, but the present results show that the approach can be used to study gene function and requirement in this important pathogen.


Subject(s)
Anti-Bacterial Agents/pharmacology , Antisense Elements (Genetics)/pharmacology , Peptide Nucleic Acids/pharmacology , Staphylococcus aureus/drug effects , Anti-Bacterial Agents/chemistry , Antisense Elements (Genetics)/genetics , Bacterial Proteins/genetics , Down-Regulation/genetics , Gene Expression/drug effects , Genes, Bacterial , Genes, Reporter/genetics , Green Fluorescent Proteins/analysis , Green Fluorescent Proteins/genetics , Peptide Nucleic Acids/genetics , RNA, Messenger/analysis , Staphylococcus aureus/genetics , Staphylococcus aureus/growth & development
10.
FASEB J ; 18(2): 394-6, 2004 Feb.
Article in English | MEDLINE | ID: mdl-14656995

ABSTRACT

Antimicrobial drug action is limited by both microbial and host cell membranes. Microbes stringently exclude the entry of most drugs, and mammalian membranes limit drug distribution and access to intracellular pathogens. Recently, cell-penetrating peptides (CPPs) have been developed as carriers to improve mammalian cell uptake. Given that CPPs are cationic and often amphipathic, similar to membrane active antimicrobial peptides, it may be possible to use CPP activity to improve drug delivery to microbes. Here, two CPPs, TP10 and pVEC, were found to enter a range of bacteria and fungi. The uptake route involves rapid surface accumulation within minutes followed by cell entry. TP10 inhibited Candida albicans and Staphylococcus aureus growth, and pVEC inhibited Mycobacterium smegmatis growth at low micromolar doses, below the levels that harmed human HeLa cells. Therefore, although TP10 and pVEC entered all cell types tested, they preferentially damage microbes, and this effect was sufficient to clear HeLa cell cultures from noninvasive S. aureus infection. Also, conversion of the cytotoxicity indicator dye SYTOX Green showed that TP10 causes rapid and lethal permeabilization of S. aureus and pVEC permeabilizes M. smegmatis, but not HeLa cells. Therefore, TP10 and pVEC can enter both mammalian and microbial cells and preferentially permeabilize and kill microbes.


Subject(s)
Anti-Infective Agents/metabolism , Cells/metabolism , Peptides/metabolism , Anti-Infective Agents/pharmacology , Bacteria/cytology , Bacteria/drug effects , Bacteria/metabolism , Cell Division/drug effects , Cell Membrane/drug effects , Cell Membrane/metabolism , Cell Membrane Permeability , Cells/cytology , Cells/drug effects , Fungi/cytology , Fungi/drug effects , Fungi/metabolism , HeLa Cells , Humans , Peptides/pharmacology , Species Specificity
11.
Biotechniques ; 35(5): 1060-4, 2003 Nov.
Article in English | MEDLINE | ID: mdl-14628680

ABSTRACT

Antibiotics are widely useful in medicine, agriculture, and industrial fermentations. However, increasing problems with resistant strains call for restrained use and alternative strategies. Antisense peptide nucleic acids (PNAs) show potent bactericidal effects when targeted against the essential Escherichia coli acpP gene. Aside from attractive antimicrobial therapeutic possibilities for such antisense PNAs, we considered that they could be used as a substitute for antibiotics in bacterial strain selection. Here, treatment of a mixture of E. coli wild-type cells and cells carrying a binding-site altered copy of acpP (acpP-1) with anti-acpP PNA completely killed wild-type cells within 2 h, whereas cells carrying acpP-1 proliferated. Furthermore, electrotransformation of E. coli cells with the plasmid carrying acpP-1 followed by PNA selection gave rise to only true transformants. Unlike previous antibiotic-free selection strategies, this procedure does not require special growth environments or special host strains. Also, the PNA-selected cells grow at a near normal rate. The results open possibilities to use antisense PNAs for strain selection and construction in research and industrial application.


Subject(s)
Cell Separation/methods , DNA, Antisense/genetics , Escherichia coli/genetics , Escherichia coli/isolation & purification , Gene Expression Regulation, Bacterial/genetics , Nucleic Acid Hybridization/methods , Peptide Nucleic Acids/genetics , Anti-Bacterial Agents/pharmacology , Escherichia coli/classification , Escherichia coli/drug effects , Gene Silencing , Species Specificity
12.
Biochem Biophys Res Commun ; 301(2): 529-34, 2003 Feb 07.
Article in English | MEDLINE | ID: mdl-12565894

ABSTRACT

Microorganisms possess stringent cell membranes which limit the cellular uptake of antimicrobials. One strategy to overcome these barriers is to attach drugs or research reagents to carrier peptides that enter cells by passive permeation or active uptake. Here the short endocytosis signal peptide NPFSD was found to efficiently deliver both FITC and GFP into Saccharomyces cerevisiae and Candida albicans with uptake into the majority of cells in a population. The NPFSD signal is itself non-toxic, but when fused to the ricin A chain toxin (RTA) the peptide enhanced both cell uptake and toxicity against C. albicans, which like other yeasts is resistant to naked RTA. Cell entry required at least 1 h incubation, temperatures above 4 degrees C, and an energy source, and uptake was out-competed with free peptide. Therefore, the NPFSD peptide can carry a range of compounds into yeasts and this delivery route holds promise to enhance the activity of antifungals.


Subject(s)
Candida albicans/metabolism , Protein Sorting Signals , Ricin/metabolism , Amino Acid Sequence , Antifungal Agents/metabolism , Antifungal Agents/toxicity , Candida albicans/drug effects , Endocytosis/physiology , Fluorescein-5-isothiocyanate/metabolism , Fluorescent Dyes/metabolism , Green Fluorescent Proteins , Indicators and Reagents/metabolism , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Microscopy, Fluorescence , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Recombinant Fusion Proteins/toxicity , Ricin/genetics , Ricin/toxicity , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/metabolism
13.
FEMS Microbiol Lett ; 215(2): 267-72, 2002 Oct 08.
Article in English | MEDLINE | ID: mdl-12399045

ABSTRACT

Stringent microbial cell barriers limit the application of many substances in research and therapeutics. Carrier peptides that penetrate or translocate across cell membranes may help overcome this problem. To assess peptide-mediated delivery into two yeast and three bacterial species, a range of cell penetrating and signal peptide sequences were fused to green fluorescent protein (GFP), expressed in Escherichia coli, partially purified and incubated with growing cells. Fluorescence microscopy indicated several peptides that mediated delivery. In particular, VLTNENPFSDP efficiently delivered GFP into Candida albicans and Staphylococcus aureus, while YKKSNNPFSD was most efficient for Bacillus subtilis and CFFKDEL for Escherichia coli. Carrier peptides may improve delivery of certain large molecular mass molecules into microorganisms for research and therapeutic applications.


Subject(s)
Bacteria/metabolism , Candida albicans/metabolism , Genetic Vectors , Luminescent Proteins/genetics , Protein Sorting Signals/genetics , Saccharomyces cerevisiae/metabolism , Amino Acid Sequence , Bacteria/genetics , Base Sequence , Candida albicans/genetics , Green Fluorescent Proteins , Image Processing, Computer-Assisted , Luminescent Proteins/metabolism , Microscopy, Fluorescence , Molecular Sequence Data , Recombinant Fusion Proteins , Saccharomyces cerevisiae/genetics
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