ABSTRACT
Amplification of the CCNE1 locus on chromosome 19q12 is prevalent in multiple tumour types, particularly in high-grade serous ovarian cancer, uterine tumours and gastro-oesophageal cancers, where high cyclin E levels are associated with genome instability, whole-genome doubling and resistance to cytotoxic and targeted therapies1-4. To uncover therapeutic targets for tumours with CCNE1 amplification, we undertook genome-scale CRISPR-Cas9-based synthetic lethality screens in cellular models of CCNE1 amplification. Here we report that increasing CCNE1 dosage engenders a vulnerability to the inhibition of the PKMYT1 kinase, a negative regulator of CDK1. To inhibit PKMYT1, we developed RP-6306, an orally bioavailable and selective inhibitor that shows single-agent activity and durable tumour regressions when combined with gemcitabine in models of CCNE1 amplification. RP-6306 treatment causes unscheduled activation of CDK1 selectively in CCNE1-overexpressing cells, promoting early mitosis in cells undergoing DNA synthesis. CCNE1 overexpression disrupts CDK1 homeostasis at least in part through an early activation of the MMB-FOXM1 mitotic transcriptional program. We conclude that PKMYT1 inhibition is a promising therapeutic strategy for CCNE1-amplified cancers.
Subject(s)
Cyclin E , Membrane Proteins , Ovarian Neoplasms , Protein Serine-Threonine Kinases , Protein-Tyrosine Kinases , CDC2 Protein Kinase , Cyclin E/genetics , Female , Gene Amplification , Gene Expression Regulation, Neoplastic , Humans , Membrane Proteins/genetics , Neoplasms/genetics , Ovarian Neoplasms/pathology , Protein Serine-Threonine Kinases/genetics , Protein-Tyrosine Kinases/genetics , Synthetic Lethal MutationsABSTRACT
The discovery of non-symmetric thienoimidazole-containing HCV NS5A inhibitors is described. The inhibitors herein reported display high potencies against both genotype 1a and 1b. In this follow-up manuscript, we discuss the importance of the linker aromaticity to achieve high potency, particularly against genotype 1a.
Subject(s)
Antiviral Agents/pharmacology , Hepacivirus/drug effects , Imidazoles/pharmacology , Viral Nonstructural Proteins/drug effects , Animals , Antiviral Agents/chemistry , Genotype , Hepacivirus/genetics , Humans , Imidazoles/chemistry , RatsABSTRACT
The discovery of C2-symmetric bis-thienoimidazoles HCV NS5A inhibitors is herein reported. Two straightforward approaches to access the requisite diyne and biphenyl linker moieties are described. This study revealed the paramount importance of the aromatic character of the linker to achieve high genotype 1a potency.
Subject(s)
Antiviral Agents/pharmacology , Drug Discovery , Imidazoles/pharmacology , Viral Nonstructural Proteins/antagonists & inhibitors , Antiviral Agents/chemistry , Imidazoles/chemistryABSTRACT
Inhibitors of the HCV NS5A nonstructural protein are showing promising clinical potential in the treatment of hepatitis C when used in combination with other direct-acting antiviral agents. Current NS5A clinical candidates such as daclatasvir, ledipasvir, and ombitasvir share a common pharmacophore that features a pair of (S)-methoxycarbonylvaline capped pyrrolidines linked to various cores by amides, imidazoles and/or benzimidazoles. In this Letter, we describe the evaluation of NS5A inhibitors which contain alternative heteroaromatic replacements for these amide mimetics. The SAR knowledge gleaned in the optimization of scaffolds containing benzoxazoles was parlayed toward the identification of potent NS5A inhibitors containing other heteroaromatic replacements such as indoles and imidazopyridines.
Subject(s)
Antiviral Agents/chemical synthesis , Antiviral Agents/pharmacology , Hepacivirus/drug effects , Viral Nonstructural Proteins/antagonists & inhibitors , Antiviral Agents/chemistry , Structure-Activity RelationshipABSTRACT
The treatment of HCV with highly efficacious, well-tolerated, interferon-free regimens is a compelling clinical goal. Trials employing combinations of direct-acting antivirals that include NS5A inhibitors have shown significant promise in meeting this challenge. Herein, we describe our efforts to identify inhibitors of NS5A and report on the discovery of benzimidazole-containing analogs with subnanomolar potency against genotype 1a and 1b replicons. Our SAR exploration of 4-substituted pyrrolidines revealed that the subtle inclusion of a 4-methyl group could profoundly increase genotype 1a potency in multiple scaffold classes.
Subject(s)
Antiviral Agents/pharmacology , Benzimidazoles/pharmacology , Pyrrolidines/pharmacology , Viral Nonstructural Proteins/drug effects , Antiviral Agents/chemistry , Benzimidazoles/chemistry , Genotype , Pyrrolidines/chemistryABSTRACT
Glycopeptides whose aminosugars have been modified by attachment of hydrophobic side chains are frequently active against vancomycin-resistant microorganisms. We have compared the conformations of six such fluorinated glycopeptides (with side chains of varying length) complexed to cell walls labeled with d-[1-(13)C]alanine, [1-(13)C]glycine, and l-[ε-(15)N]lysine in whole cells of Staphylococcus aureus. The internuclear distances from (19)F of the bound drug to the (13)C and (15)N labels of the peptidoglycan, and to the natural abundance (31)P of lipid membranes and teichoic acids, were determined by rotational-echo double resonance NMR. The drugs did not dimerize, and their side chains did not form membrane anchors but instead became essential parts of secondary binding to pentaglycyl bridge segments of the cell-wall peptidoglycan.
Subject(s)
Glycopeptides/chemistry , Glycopeptides/metabolism , Peptidoglycan/chemistry , Staphylococcus aureus/metabolism , Alanine/genetics , Binding Sites , Cell Membrane/metabolism , Glycine/genetics , Hydrophobic and Hydrophilic Interactions , Lysine/genetics , Nuclear Magnetic Resonance, Biomolecular , Peptidoglycan/metabolismABSTRACT
PKMYT1 is a regulator of CDK1 phosphorylation and is a compelling therapeutic target for the treatment of certain types of DNA damage response cancers due to its established synthetic lethal relationship with CCNE1 amplification. To date, no selective inhibitors have been reported for this kinase that would allow for investigation of the pharmacological role of PKMYT1. To address this need compound 1 was identified as a weak PKMYT1 inhibitor. Introduction of a dimethylphenol increased potency on PKMYT1. These dimethylphenol analogs were found to exist as atropisomers that could be separated and profiled as single enantiomers. Structure-based drug design enabled optimization of cell-based potency. Parallel optimization of ADME properties led to the identification of potent and selective inhibitors of PKMYT1. RP-6306 inhibits CCNE1-amplified tumor cell growth in several preclinical xenograft models. The first-in-class clinical candidate RP-6306 is currently being evaluated in Phase 1 clinical trials for treatment of various solid tumors.
Subject(s)
Neoplasms , Protein-Tyrosine Kinases , Cell Line, Tumor , Cell Proliferation , Humans , Membrane Proteins , Neoplasms/pathology , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Protein Serine-Threonine KinasesABSTRACT
As therapeutic agents of choice in the treatment of complicated infections, glycopeptide antibiotics are often preferentially used in cases of osteomyelitis, an infection located in bone and notoriously difficult to successfully manage. Yet frequent and heavy doses of these systemically administered antibiotics are conventionally prescribed to obtain higher antibiotic levels in the bone and reduce the high recurrence rates. Targeting antibiotics to the bone after systemic administration would present at least three potential advantages: (i) greater efficacy, by concentrating the therapeutic agent in bone; (ii) greater convenience, through a reduction in the frequency of administration; and (iii) greater safety, by reducing the levels of systemic drug exposure. We present here the design, synthesis and in vitro evaluation of eight prodrugs of the glycopeptide antibacterial agents vancomycin and oritavancin taking advantage of the affinity of the bisphosphonate group for bone for delivery to osseous tissues.
Subject(s)
Diphosphonates/chemical synthesis , Glycopeptides/chemical synthesis , Osteomyelitis/drug therapy , Prodrugs/chemical synthesis , Animals , Anti-Bacterial Agents/pharmacology , Bone Density Conservation Agents/chemical synthesis , Bone Density Conservation Agents/chemistry , Bone Density Conservation Agents/therapeutic use , Diphosphonates/chemistry , Diphosphonates/therapeutic use , Glycopeptides/chemistry , Glycopeptides/therapeutic use , Humans , Lipoglycopeptides , Mice , Microbial Sensitivity Tests , Molecular Structure , Prodrugs/chemistry , Prodrugs/therapeutic use , Rats , Staphylococcus aureus/drug effects , Vancomycin/therapeutic useABSTRACT
Osteomyelitis is a difficult to treat bacterial infection of the bone. Delivering antibacterial agents to the bone may overcome the difficulties in treating this illness by effectively concentrating the antibiotic at the site of infection and by limiting the toxicity that may result from systemic exposure to the large doses conventionally used. Using bisphosphonates as osteophilic functional groups, different forms of fluoroquinolone esters were synthesized and evaluated for their ability to bind bone and to release the parent antibacterial agent. Bisphosphonated glycolamide fluoroquinolone esters were found to present a profile consistent with effective and rapid bone binding and efficient release of the active drug moiety. They were assessed for their ability to prevent bone infection in vivo and were found to be effective when the free fluoroquinolones were not.
Subject(s)
Esters/chemical synthesis , Esters/pharmacology , Fluoroquinolones/chemistry , Organophosphonates/chemistry , Osteomyelitis/prevention & control , Prodrugs/chemical synthesis , Prodrugs/pharmacology , Animals , Disease Models, Animal , Esters/chemistry , Female , Humans , Molecular Structure , Prodrugs/chemistry , RatsABSTRACT
The increasing frequency of Enterococcus faecium isolates with multidrug resistance is a serious clinical problem given the severely limited number of therapeutic options available to treat these infections. Oritavancin is a promising new alternative in clinical development that has potent antimicrobial activity against both staphylococcal and enterococcal vancomycin-resistant pathogens. Using solid-state NMR to detect changes in the cell-wall structure and peptidoglycan precursors of whole cells after antibiotic-induced stress, we report that vancomycin and oritavancin have different modes of action in E. faecium. Our results show the accumulation of peptidoglycan precursors after vancomycin treatment, consistent with transglycosylase inhibition, but no measurable difference in cross-linking. In contrast, after oritavancin exposure, we did not observe the accumulation of peptidoglycan precursors. Instead, the number of cross-links is significantly reduced, showing that oritavancin primarily inhibits transpeptidation. We propose that the activity of oritavancin is the result of a secondary binding interaction with the E. faecium peptidoglycan. The hypothesis is supported by results from (13)C{(19)F} rotational-echo double-resonance (REDOR) experiments on whole cells enriched with l-[1-(13)C]lysine and complexed with desleucyl [(19)F]oritavancin. These experiments establish that an oritavancin derivative with a damaged d-Ala-d-Ala binding pocket still binds to E. faecium peptidoglycan. The (13)C{(19)F} REDOR dephasing maximum indicates that the secondary binding site of oritavancin is specific to nascent and template peptidoglycan. We conclude that the inhibition of transpeptidation by oritavancin in E. faecium is the result of the large number of secondary binding sites relative to the number of primary binding sites.
Subject(s)
Anti-Bacterial Agents/pharmacology , Enterococcus faecium/drug effects , Glycopeptides/pharmacology , Vancomycin/pharmacology , Carbon Isotopes/metabolism , Cell Wall/chemistry , Cell Wall/drug effects , Enterococcus faecium/chemistry , Fluorine/metabolism , Lipoglycopeptides , Magnetic Resonance Spectroscopy/methods , Molecular Structure , Peptidoglycan/metabolismABSTRACT
Solid-state NMR measurements performed on intact whole cells of Staphylococcus aureus labeled selectively in vivo have established that des-N-methylleucyl oritavancin (which has antimicrobial activity) binds to the cell-wall peptidoglycan, even though removal of the terminal N-methylleucyl residue destroys the D-Ala-D-Ala binding pocket. By contrast, the des-N-methylleucyl form of vancomycin (which has no antimicrobial activity) does not bind to the cell wall. Solid-state NMR has also determined that oritavancin and vancomycin are comparable inhibitors of transglycosylation, but that oritavancin is a more potent inhibitor of transpeptidation. This combination of effects on cell-wall binding and biosynthesis is interpreted in terms of a recent proposal that oritavancin-like glycopeptides have two cell-wall binding sites: the well-known peptidoglycan D-Ala-D-Ala pentapeptide stem terminus and the pentaglycyl bridging segment. The resulting dual mode of action provides a structural framework for coordinated cell-wall assembly that accounts for the enhanced potency of oritavancin and oritavancin-like analogues against vancomycin-resistant organisms.
Subject(s)
Anti-Bacterial Agents/pharmacology , Cell Wall/drug effects , Cell Wall/metabolism , Glycopeptides/pharmacology , Staphylococcus aureus/cytology , Staphylococcus aureus/drug effects , Amino Acids/metabolism , Anti-Bacterial Agents/metabolism , Carbon Isotopes , Cross-Linking Reagents/pharmacology , Fluorine , Glycopeptides/metabolism , Lipoglycopeptides , Magnetic Resonance Spectroscopy , Microbial Sensitivity Tests , Models, Biological , Models, Molecular , Nitrogen Isotopes , Peptidoglycan/biosynthesis , Peptidoglycan/chemistryABSTRACT
Osteomyelitis is an infection located in bone and a notoriously difficult disease to manage, requiring frequent and heavy doses of systemically administered antibiotics. Targeting antibiotics to the bone after systemic administration may provide both greater efficacy of treatment and less frequent administration. By taking advantage of the affinity of the bisphosphonate group for bone mineral, we have prepared a set of 13 bisphosphonated antibacterial prodrugs based on eight different linkers tethered to the free amino functionality on fluoroquinolone antibiotics. While all but one of the prodrugs were shown in vitro to be effective and rapid bone binders (over 90% in 1 h), only eight of them demonstrated the capacity to significantly regenerate the parent drug. In a rat model of the disease, a selected group of agents demonstrated their ability to prevent osteomyelitis when used in circumstances under which the parent drug had already been cleared and is thus inactive.
Subject(s)
Diphosphonates/chemistry , Diphosphonates/pharmacology , Fluoroquinolones/chemical synthesis , Fluoroquinolones/pharmacology , Osteomyelitis/prevention & control , Prodrugs/chemical synthesis , Prodrugs/pharmacology , Amines/chemistry , Animals , Cell Line , Female , Fluoroquinolones/chemistry , Molecular Structure , Prodrugs/chemistry , Rats , Structure-Activity RelationshipABSTRACT
The preparation and biological evaluation of 5-substituted-6-hydroxy-2-(anilino)pyrimidinones as a new class of DNA polymerase IIIC inhibitors, required for the replication of chromosomal DNA in Gram-positive bacteria, are described. These new dGTP competitive inhibitors displayed good levels of in vitro inhibition and antibacterial activity against Staphylococcus aureus. A new class of dATP competitive inhibitors, 6-substituted-2-amino-5-alkyl-pyrimidin-4-ones, whose antibacterial activity was unaffected by serum, were identified.
Subject(s)
DNA Polymerase III/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Pyrimidinones/pharmacology , Staphylococcus aureus/enzymology , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Microbial Sensitivity Tests , Molecular Structure , Pyrimidinones/chemical synthesis , Pyrimidinones/chemistry , Staphylococcus aureus/drug effects , Structure-Activity RelationshipABSTRACT
The RNA polymerase holoenzyme is a proven target for antibacterial agents. A high-throughput screening program based on this enzyme from Staphylococcus aureus had previously identified a 2-ureidothiophene-3-carboxylate as a low micromolar inhibitor. An investigation of the relationships between the structures of this class of compounds and their inhibitory- and antibacterial activities is described here, leading to a set of potent RNA polymerase inhibitors with antibacterial activity. Characterization of this bioactivity, including studies of the mechanism of action, is provided, highlighting the power of the reverse chemical genetics approach in providing tools to inhibit the bacterial RNA polymerase.
Subject(s)
Anti-Bacterial Agents/classification , Carboxylic Acids/chemistry , Carboxylic Acids/pharmacology , DNA-Directed RNA Polymerases/antagonists & inhibitors , Drug Resistance, Bacterial , Rifampin/pharmacology , Staphylococcus aureus/drug effects , Thiophenes/chemistry , Thiophenes/pharmacology , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , DNA-Directed RNA Polymerases/metabolism , Molecular Structure , Molecular Weight , Structure-Activity RelationshipABSTRACT
Enantiopure (3S,5R,8S)-3-[N-(Boc)amino]-1-azabicyclo[3.3.0]octan-2-one 8-carboxylic acid (1) was synthesized in nine steps and 16% overall yield from aspartate beta-aldehyde 7. Carbene-catalyzed acyloin condensation of 7, followed by acetylation and samarium iodide reduction, gave linear precursor (2S,7S)-alpha,omega-diamino-4-oxosuberate 11, which was converted to N-(Boc)aminopyrrolizidin-2-one carboxylic acid 1 by a reductive amination/lactam cyclization sequence. X-ray analysis of (3S,5R,8S)-methyl N-(Boc)aminopyrrolizidin-2-one carboxylate 21 showed that its internal backbone dihedral angles (psi = -149 degrees, phi = -49 degrees ) were in good agreement with the ideal values for a type II' beta-turn. Proton NMR experiments on N'-methyl-N-(Boc)aminopyrrolizidin-2-one carboxamide 23 demonstrated significantly different NH chemical displacements and temperature coefficients suggestive of solvent shielded and exposed hydrogens indicative of a turn conformation. Because pyrrolizidinone amino acids can serve as conformationally rigid dipeptide surrogates, this synthesis should facilitate their application in the exploration of conformation-activity relationships of various biologically active peptides.