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
Pathogenic microbes have evolved complex secretion systems to deliver virulence factors into host cells. Identification of these factors is critical for understanding the infection process. We report a powerful and versatile approach to the selective labeling and identification of secreted pathogen proteins. Selective labeling of microbial proteins is accomplished via translational incorporation of azidonorleucine (Anl), a methionine surrogate that requires a mutant form of the methionyl-tRNA synthetase for activation. Secreted pathogen proteins containing Anl can be tagged by azide-alkyne cycloaddition and enriched by affinity purification. Application of the method to analysis of the type III secretion system of the human pathogen Yersinia enterocolitica enabled efficient identification of secreted proteins, identification of distinct secretion profiles for intracellular and extracellular bacteria, and determination of the order of substrate injection into host cells. This approach should be widely useful for the identification of virulence factors in microbial pathogens and the development of potential new targets for antimicrobial therapy.
Subject(s)
Amino Acids/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Microbiological Techniques , Anti-Bacterial Agents/chemistry , HeLa Cells , Humans , Mass Spectrometry , Methionine-tRNA Ligase/chemistry , Microscopy, Confocal , Microscopy, Fluorescence , Models, Biological , Mutation , Norleucine/chemistry , Proteome , Proteomics/methods , Time Factors , Virulence Factors , Yersinia enterocolitica/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
The azide-alkyne cycloaddition provides a powerful tool for bio-orthogonal labeling of proteins, nucleic acids, glycans, and lipids. In some labeling experiments, e.g., in proteomic studies involving affinity purification and mass spectrometry, it is convenient to use cleavable probes that allow release of labeled biomolecules under mild conditions. Five cleavable biotin probes are described for use in labeling of proteins and other biomolecules via azide-alkyne cycloaddition. Subsequent to conjugation with metabolically labeled protein, these probes are subject to cleavage with either 50 mM Na(2)S(2)O(4), 2% HOCH(2)CH(2)SH, 10% HCO(2)H, 95% CF(3)CO(2)H, or irradiation at 365 nm. Most strikingly, a probe constructed around a dialkoxydiphenylsilane (DADPS) linker was found to be cleaved efficiently when treated with 10% HCO(2)H for 0.5 h. A model green fluorescent protein was used to demonstrate that the DADPS probe undergoes highly selective conjugation and leaves a small (143 Da) mass tag on the labeled protein after cleavage. These features make the DADPS probe especially attractive for use in biomolecular labeling and proteomic studies.
Subject(s)
Alkynes/chemistry , Azides/chemistry , Biotin/chemistry , Molecular Probes/chemistry , Proteins/chemistry , Cyclization , Dapsone , Green Fluorescent Proteins/chemistry , Molecular Probes/chemical synthesis , Protein Structure, SecondaryABSTRACT
Incorporation of an hydrophobic (phenethylamino)ethyl ether at C2â³ of N1-(HABA)-3',4'-dideoxyparomomycin led to a novel analog with an excellent antibacterial profile against a host of resistant bacteria.
Subject(s)
Aminoglycosides/chemistry , Anti-Bacterial Agents/chemistry , Bacteria/drug effects , Drug Design , RNA, Ribosomal/chemistry , Anti-Bacterial Agents/pharmacology , Crystallization , Drug Resistance, Bacterial , Hydrophobic and Hydrophilic InteractionsABSTRACT
A series of 2"-O-substituted ether analogues of paromomycin were prepared based on new site-selective functionalizations. X-ray cocrystal complexes of several such analogues revealed a new mode of binding in the A-site rRNA, whereby rings I and II adopted the familiar orientation and position previously observed with paromomycin, but rings III and IV were oriented differently. With few exceptions, all of the new analogues showed potent inhibitory activity equal or better than paromomycin against a sensitive strain of S. aureus. Single digit microM MIC values were obtained against E. coli, with some of the ether appendages containing polar or basic end groups. Two analogues showed excellent survival rate in a mouse septicemia protection assay. Preliminary histopathological analysis of the kidney showed no overt signs of toxicity, while controls with neomycin and kanamycin were toxic at lower doses.
Subject(s)
Anti-Bacterial Agents/chemical synthesis , Paromomycin/analogs & derivatives , Paromomycin/chemical synthesis , RNA, Ribosomal/chemistry , Animals , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Binding Sites , Crystallography, X-Ray , Drug Design , Escherichia coli/drug effects , Ethers/chemical synthesis , Ethers/chemistry , Ethers/pharmacology , Female , Mice , Mice, Inbred BALB C , Microbial Sensitivity Tests , Models, Molecular , Molecular Structure , Paromomycin/chemistry , Paromomycin/pharmacology , Sepsis/prevention & control , Staphylococcal Infections/prevention & control , Staphylococcus aureus/drug effects , Structure-Activity RelationshipABSTRACT
This review brings to the forefront key synthetic modifications on natural products (NPs) that have yielded successful drugs. The emphasis is placed on the power of targeted chemical transformations in enhancing the therapeutic value of NPs through optimization of pharmacokinetics, stability, potency, and/or selectivity. Multiple classes of NPs such as macrolides, opioids, steroids, and ß-lactams used to treat a variety of conditions such as cancers, infections, inflammation are exemplified. Molecular modeling or X-ray structures of NP/protein complexes supporting the observed boost in therapeutic value of the modified NPs are also discussed. Significant advancement in synthetic chemistry, in structure determination, and in the understanding of factors controlling pharmacokinetics can now better position drug discovery teams to undertake NPs as valuable leads. We hope that the beneficial NPs synthetic modifications outlined here will reignite medicinal chemists' interest in NPs and their derivatives.
Subject(s)
Biological Products/chemistry , Drug Design , Analgesics, Opioid/chemistry , Animals , Anti-Bacterial Agents/chemistry , Chemistry Techniques, Synthetic , Crystallography, X-Ray , Drug Discovery , Humans , Macrolides/chemistry , Models, Molecular , Molecular Structure , Neoplasms/drug therapy , Peptides/chemistry , Solubility , Solvents , Steroids/chemistry , Structure-Activity Relationship , Taxoids/chemistrySubject(s)
Anti-Bacterial Agents/metabolism , Anti-Bacterial Agents/pharmacology , Paromomycin/metabolism , Paromomycin/pharmacology , RNA, Ribosomal/metabolism , Anti-Bacterial Agents/chemistry , Models, Molecular , Molecular Conformation , Molecular Structure , Paromomycin/chemistry , RNA, Ribosomal/genetics , Structure-Activity RelationshipABSTRACT
A synthesis of verdamicin C2 and its congener C2a has been accomplished from sisomicin relying on a novel oxidative transformation of an allylic azide to the corresponding alpha,beta-unsaturated aldehyde, and its stereocontrolled elaboration into the intended 5' side chain of verdamicin C2 and C2a. In vitro antibacterial testing shows that both C6' epimers in verdamicin C2 and C2a are equally active against a variety of bacterial strains. Oxidation of allylic primary azides, ethers, and esters of 2-substituted dihydro[2H]pyrans with SeO(2) leads directly to the corresponding aldehydes.
Subject(s)
Aminoglycosides/chemical synthesis , Aminoglycosides/pharmacology , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/pharmacology , Azides/chemistry , Pyrans/chemistry , Aminoglycosides/chemistry , Aminoglycosides/toxicity , Animals , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/toxicity , Bacteria/drug effects , Mice , Oxidation-ReductionABSTRACT
Based on molecular modeling and available X-ray structure data on aminoglycosides complexed with a bacterial ribosomal surrogate or with a kinase, two analogues of paromomycin were prepared by tethering the 6-OH and the 6'''-NH(2) group with a five-carbon bridge. Only one of two possible hydroxyl groups was phosphorylated by the kinase. The application of ring closure metathesis is presented for the first time to construct bridged macrocyclic analogues in the aminoglycoside series.
Subject(s)
Aminoglycosides/chemistry , Kanamycin Kinase/chemistry , Molecular Probes/chemistry , Aminoglycosides/pharmacology , Escherichia coli/drug effects , Models, Molecular , Molecular Structure , Staphylococcus aureus/drug effectsABSTRACT
The crystal structure of the complex between oligonucleotide containing the bacterial ribosomal decoding site (A site) and the synthetic paromomycin analogue 1, which contains the gamma-amino-alpha-hydroxybutyryl (L-haba) group at position N1 of ring II (2-DOS ring), and an ether chain with an O-phenethylaminoethyl group at position C2'' of ring III, is reported. Interestingly, next to the paromomycin analogue 1 specifically bound to the A site, a second molecule of 1 with a different conformation is observed at the crystal packing interface which mimics the A-minor interaction between two bulged-out adenines from the A site and the codon-anticodon stem of the mRNA-tRNA complex. Improved antibacterial activity supports the conclusion that analogue 1 might affect protein synthesis on the ribosome in two different ways: 1) specific binding to the A site forces maintenance of the "on" state with two bulged out adenines, and 2) a new binding mode of 1 to an A-minor motif which stabilizes complex formation between the ribosome and the mRNA-tRNA complex regardless of whether the codon-anticodon stem is of the cognate or near-cognate type.