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1.
bioRxiv ; 2024 May 15.
Article in English | MEDLINE | ID: mdl-38952800

ABSTRACT

Cyclin-dependent kinase 9 (CDK9) coordinates signaling events that regulate RNA polymerase II (Pol II) pause-release states. It is an important co-factor for transcription factors, such as MYC, that drive aberrant cell proliferation when their expression is deregulated. CDK9 modulation offers an approach for attenuating dysregulation in such transcriptional programs. As a result, numerous drug development campaigns to inhibit CDK9 kinase activity have been pursued. More recently, targeted degradation has emerged as an attractive approach. However, comprehensive evaluation of degradation versus inhibition is still critically needed to assess the biological contexts in which degradation might offer superior therapeutic benefits. We validated that CDK9 inhibition triggers a compensatory mechanism that dampens its effect on MYC expression and found that this feedback mechanism was absent when the kinase is degraded. Importantly, CDK9 degradation is more effective than its inhibition for disrupting MYC transcriptional regulatory circuitry likely through the abrogation of both enzymatic and scaffolding functions of CDK9. Highlights: - KI-CDK9d-32 is a highly potent and selective CDK9 degrader. - KI-CDK9d-32 leads to rapid downregulation of MYC protein and mRNA transcripts levels. - KI-CDK9d-32 represses canonical MYC pathways and leads to a destabilization of nucleolar homeostasis. - Multidrug resistance ABCB1 gene emerged as the strongest resistance marker for the CDK9 PROTAC degrader.

2.
Nat Commun ; 15(1): 317, 2024 Jan 05.
Article in English | MEDLINE | ID: mdl-38182597

ABSTRACT

Thermus thermophilus bacteriophage P23-45 encodes a giant 5,002-residue tail tape measure protein (TMP) that defines the length of its extraordinarily long tail. Here, we show that the N-terminal portion of P23-45 TMP is an unusual RNA polymerase (RNAP) homologous to cellular RNAPs. The TMP-fused virion RNAP transcribes pre-early phage genes, including a gene that encodes another, non-virion RNAP, that transcribes early and some middle phage genes. We report the crystal structures of both P23-45 RNAPs. The non-virion RNAP has a crab-claw-like architecture. By contrast, the virion RNAP adopts a unique flat structure without a clamp. Structure and sequence comparisons of the P23-45 RNAPs with other RNAPs suggest that, despite the extensive functional differences, the two P23-45 RNAPs originate from an ancient gene duplication in an ancestral phage. Our findings demonstrate striking adaptability of RNAPs that can be attained within a single virus species.


Subject(s)
Bacteriophages , Pyridinolcarbamate , Virion/genetics , Bacteriophages/genetics , Bacterial Typing Techniques , DNA-Directed RNA Polymerases/genetics
3.
Biochemistry ; 61(15): 1633-1641, 2022 08 02.
Article in English | MEDLINE | ID: mdl-35856337

ABSTRACT

Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to the corresponding deoxyribonucleotides. The catalytic activity of most RNRs depends on the formation of a dimer of the catalytic subunits. The active site is located at the interface, and part of the substrate binding site and regulatory mechanisms work across the subunit in the dimer. In this study, we describe and characterize a novel domain responsible for forming the catalytic dimer in several class II RNRs. The 3D structure of the class II RNR from Rhodobacter sphaeroides reveals a so far undescribed α-helical domain in the dimer interface, which is embracing the other subunit. Genetic removal of this HUG domain leads to a severe reduction of activity paired with reduced dimerization capability. In comparison with other described RNRs, the enzyme with this domain is less dependent on the presence of nucleotides to act as allosteric effectors in the formation of dimers. The HUG domain appears to serve as an interlock to keep the dimer intact and functional even at low enzyme and/or effector concentrations.


Subject(s)
Ribonucleotide Reductases , Allosteric Regulation , Binding Sites , Catalytic Domain , Models, Molecular , Ribonucleotide Reductases/chemistry
4.
Nat Struct Mol Biol ; 28(9): 740-746, 2021 09.
Article in English | MEDLINE | ID: mdl-34381216

ABSTRACT

Molnupiravir is an orally available antiviral drug candidate currently in phase III trials for the treatment of patients with COVID-19. Molnupiravir increases the frequency of viral RNA mutations and impairs SARS-CoV-2 replication in animal models and in humans. Here, we establish the molecular mechanisms underlying molnupiravir-induced RNA mutagenesis by the viral RNA-dependent RNA polymerase (RdRp). Biochemical assays show that the RdRp uses the active form of molnupiravir, ß-D-N4-hydroxycytidine (NHC) triphosphate, as a substrate instead of cytidine triphosphate or uridine triphosphate. When the RdRp uses the resulting RNA as a template, NHC directs incorporation of either G or A, leading to mutated RNA products. Structural analysis of RdRp-RNA complexes that contain mutagenesis products shows that NHC can form stable base pairs with either G or A in the RdRp active center, explaining how the polymerase escapes proofreading and synthesizes mutated RNA. This two-step mutagenesis mechanism probably applies to various viral polymerases and can explain the broad-spectrum antiviral activity of molnupiravir.


Subject(s)
COVID-19/prevention & control , Cytidine/analogs & derivatives , Hydroxylamines/metabolism , Mutagenesis/genetics , RNA, Viral/genetics , SARS-CoV-2/genetics , Animals , Antiviral Agents/chemistry , Antiviral Agents/metabolism , Antiviral Agents/pharmacology , Base Sequence , COVID-19/virology , Cytidine/chemistry , Cytidine/metabolism , Cytidine/pharmacology , Humans , Hydroxylamines/chemistry , Hydroxylamines/pharmacology , Models, Molecular , Molecular Structure , Mutagenesis/drug effects , Mutation/drug effects , Mutation/genetics , Nucleic Acid Conformation , Protein Binding/drug effects , Protein Conformation , RNA, Viral/chemistry , RNA, Viral/metabolism , RNA-Dependent RNA Polymerase/chemistry , RNA-Dependent RNA Polymerase/genetics , RNA-Dependent RNA Polymerase/metabolism , SARS-CoV-2/drug effects , SARS-CoV-2/physiology , Virus Replication/drug effects , Virus Replication/genetics , COVID-19 Drug Treatment
5.
Cell Chem Biol ; 28(2): 134-147.e14, 2021 02 18.
Article in English | MEDLINE | ID: mdl-33086052

ABSTRACT

Castration-resistant prostate cancers (CRPCs) lose sensitivity to androgen-deprivation therapies but frequently remain dependent on oncogenic transcription driven by the androgen receptor (AR) and its splice variants. To discover modulators of AR-variant activity, we used a lysate-based small-molecule microarray assay and identified KI-ARv-03 as an AR-variant complex binder that reduces AR-driven transcription and proliferation in prostate cancer cells. We deduced KI-ARv-03 to be a potent, selective inhibitor of CDK9, an important cofactor for AR, MYC, and other oncogenic transcription factors. Further optimization resulted in KB-0742, an orally bioavailable, selective CDK9 inhibitor with potent anti-tumor activity in CRPC models. In 22Rv1 cells, KB-0742 rapidly downregulates nascent transcription, preferentially depleting short half-life transcripts and AR-driven oncogenic programs. In vivo, oral administration of KB-0742 significantly reduced tumor growth in CRPC, supporting CDK9 inhibition as a promising therapeutic strategy to target AR dependence in CRPC.


Subject(s)
Androgen Receptor Antagonists/pharmacology , Cyclin-Dependent Kinase 9/antagonists & inhibitors , Prostatic Neoplasms, Castration-Resistant/drug therapy , Protein Kinase Inhibitors/pharmacology , Receptors, Androgen/genetics , Transcription, Genetic/drug effects , Androgen Receptor Antagonists/therapeutic use , Animals , Cell Line, Tumor , Cyclin-Dependent Kinase 9/genetics , Gene Expression Regulation, Neoplastic/drug effects , Male , Mice , Mice, Inbred BALB C , Models, Molecular , Prostatic Neoplasms, Castration-Resistant/genetics , Protein Kinase Inhibitors/therapeutic use
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