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1.
J Chem Inf Model ; 63(21): 6696-6705, 2023 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-37831965

RESUMO

Photoswitchable (PSW) molecules offer an attractive opportunity for the optical control of biological processes. However, the successful design of such compounds remains a challenging multioptimization endeavor, resulting in several biological target classes still relatively poorly explored by photoswitchable ligands, as is the case for G protein-coupled receptors (GPCRs). Here, we present the PSW-Designer, a fully open-source computational platform, implemented in the KNIME Analytics Platform, to design and virtually screen novel photoswitchable ligands for photopharmacological applications based on privileged scaffolds. We demonstrate the applicability of the PSW-Designer to GPCRs and assess its predictive capabilities via two retrospective case studies. Furthermore, by leveraging bioactivity information on known ligands, typical and atypical strategies for photoswitchable group incorporation, and the increasingly structural information available for biological targets, the PSW-Design will facilitate the design of novel photoswitchable molecules with improved photopharmacological properties and increased binding affinity shifts upon illumination for GPCRs and many other protein targets.


Assuntos
Receptores Acoplados a Proteínas G , Estudos Retrospectivos , Receptores Acoplados a Proteínas G/química , Ligantes
2.
Nat Chem Biol ; 16(10): 1120-1128, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32690945

RESUMO

The NUDIX hydrolase NUDT15 was originally implicated in sanitizing oxidized nucleotides, but was later shown to hydrolyze the active thiopurine metabolites, 6-thio-(d)GTP, thereby dictating the clinical response of this standard-of-care treatment for leukemia and inflammatory diseases. Nonetheless, its physiological roles remain elusive. Here, we sought to develop small-molecule NUDT15 inhibitors to elucidate its biological functions and potentially to improve NUDT15-dependent chemotherapeutics. Lead compound TH1760 demonstrated low-nanomolar biochemical potency through direct and specific binding into the NUDT15 catalytic pocket and engaged cellular NUDT15 in the low-micromolar range. We also employed thiopurine potentiation as a proxy functional readout and demonstrated that TH1760 sensitized cells to 6-thioguanine through enhanced accumulation of 6-thio-(d)GTP in nucleic acids. A biochemically validated, inactive structural analog, TH7285, confirmed that increased thiopurine toxicity takes place via direct NUDT15 inhibition. In conclusion, TH1760 represents the first chemical probe for interrogating NUDT15 biology and potential therapeutic avenues.


Assuntos
Pirofosfatases/antagonistas & inibidores , Pirofosfatases/metabolismo , Sítios de Ligação , Linhagem Celular , Desenho de Fármacos , Desenvolvimento de Medicamentos , Escherichia coli , Humanos , Pirofosfatase Inorgânica/antagonistas & inibidores , Pirofosfatase Inorgânica/genética , Pirofosfatase Inorgânica/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Pirofosfatases/química , Pirofosfatases/genética , Relação Estrutura-Atividade
3.
Nucleic Acids Res ; 48(21): 12234-12251, 2020 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-33211885

RESUMO

Altered oncogene expression in cancer cells causes loss of redox homeostasis resulting in oxidative DNA damage, e.g. 8-oxoguanine (8-oxoG), repaired by base excision repair (BER). PARP1 coordinates BER and relies on the upstream 8-oxoguanine-DNA glycosylase (OGG1) to recognise and excise 8-oxoG. Here we hypothesize that OGG1 may represent an attractive target to exploit reactive oxygen species (ROS) elevation in cancer. Although OGG1 depletion is well tolerated in non-transformed cells, we report here that OGG1 depletion obstructs A3 T-cell lymphoblastic acute leukemia growth in vitro and in vivo, validating OGG1 as a potential anti-cancer target. In line with this hypothesis, we show that OGG1 inhibitors (OGG1i) target a wide range of cancer cells, with a favourable therapeutic index compared to non-transformed cells. Mechanistically, OGG1i and shRNA depletion cause S-phase DNA damage, replication stress and proliferation arrest or cell death, representing a novel mechanistic approach to target cancer. This study adds OGG1 to the list of BER factors, e.g. PARP1, as potential targets for cancer treatment.


Assuntos
Neoplasias do Colo/tratamento farmacológico , DNA Glicosilases/genética , DNA de Neoplasias/genética , Regulação Neoplásica da Expressão Gênica , Poli(ADP-Ribose) Polimerase-1/imunologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/mortalidade , Dano ao DNA , DNA Glicosilases/antagonistas & inibidores , DNA Glicosilases/metabolismo , Reparo do DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , DNA de Neoplasias/metabolismo , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Guanina/análogos & derivados , Guanina/metabolismo , Células HCT116 , Humanos , Camundongos , Camundongos Nus , Terapia de Alvo Molecular , Estresse Oxidativo , Poli(ADP-Ribose) Polimerase-1/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Análise de Sobrevida , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Nature ; 508(7495): 215-21, 2014 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-24695224

RESUMO

Cancers have dysfunctional redox regulation resulting in reactive oxygen species production, damaging both DNA and free dNTPs. The MTH1 protein sanitizes oxidized dNTP pools to prevent incorporation of damaged bases during DNA replication. Although MTH1 is non-essential in normal cells, we show that cancer cells require MTH1 activity to avoid incorporation of oxidized dNTPs, resulting in DNA damage and cell death. We validate MTH1 as an anticancer target in vivo and describe small molecules TH287 and TH588 as first-in-class nudix hydrolase family inhibitors that potently and selectively engage and inhibit the MTH1 protein in cells. Protein co-crystal structures demonstrate that the inhibitors bind in the active site of MTH1. The inhibitors cause incorporation of oxidized dNTPs in cancer cells, leading to DNA damage, cytotoxicity and therapeutic responses in patient-derived mouse xenografts. This study exemplifies the non-oncogene addiction concept for anticancer treatment and validates MTH1 as being cancer phenotypic lethal.


Assuntos
Enzimas Reparadoras do DNA/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Nucleotídeos/metabolismo , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Animais , Domínio Catalítico , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cristalização , Dano ao DNA , Enzimas Reparadoras do DNA/química , Enzimas Reparadoras do DNA/metabolismo , Nucleotídeos de Desoxiguanina/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Feminino , Humanos , Masculino , Camundongos , Modelos Moleculares , Conformação Molecular , Terapia de Alvo Molecular , Neoplasias/patologia , Oxirredução/efeitos dos fármacos , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/metabolismo , Pirimidinas/química , Pirimidinas/farmacocinética , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Pirofosfatases/antagonistas & inibidores , Reprodutibilidade dos Testes , Ensaios Antitumorais Modelo de Xenoenxerto , Nudix Hidrolases
6.
J Mol Biol ; 436(15): 168643, 2024 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-38848865

RESUMO

Autophagy facilitates the degradation of cellular content via the lysosome and is involved in cellular homeostasis and stress response pathways. As such, malfunction of autophagy is linked to a variety of diseases ranging from organ-specific illnesses like cardiomyopathy to systemic illnesses such as cancer or metabolic syndromes. Given the variety of autophagic functions within a cell and tissue, regulation of autophagy is complex and contains numerous positive and negative feedback loops. While our knowledge of mechanisms for cargo selectivity has significantly improved over the last decade, our understanding of signaling routes activating individual autophagy pathways remains rather sparse. In this resource study, we report on a well-characterized chemical library containing 77 GPCR-targeting ligands that was used to systematically analyze LC3B-based autophagy as well as ER-phagy flux upon compound treatment. Upon others, compounds TC-G 1004, BAY 60-6583, PSNCBAM-1, TC-G 1008, LPA2 Antagonist 1, ML-154, JTC-801 and ML-290 targeting adenosine receptor A2a (ADORA2A), adenosine receptor A2b (ADORA2B), cannabinoid receptor 1 (CNR1), G-protein coupled receptor 39 (GPR39), lysophosphatidic acid receptor 2 (LPAR2), neuropeptide S receptor 1 (NPSR1), opioid related nociceptin receptor 1 (OPRL1), and relaxin receptor 1 (RXFP1), respectively, were hit compounds for general autophagy flux. From these compounds, only JTC-801 markly increased ER-phagy flux. In addition, the global impact of these selected hit compounds were analyzed by TMT-based mass spectrometry and demonstrated the differential impact of targeting GPCRs on autophagy-associated proteins. This chemical screening exercise indicates to a significant cross-talk between GPCR signaling and regulation of autophagy pathways.


Assuntos
Autofagia , Receptores Acoplados a Proteínas G , Autofagia/efeitos dos fármacos , Humanos , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Ligantes
7.
iScience ; 27(2): 108907, 2024 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-38318365

RESUMO

SAMHD1 is a dNTP triphosphohydrolase governing nucleotide pool homeostasis and can detoxify chemotherapy metabolites controlling their clinical responses. To understand SAMHD1 biology and investigate the potential of targeting SAMHD1 as neoadjuvant to current chemotherapies, we set out to discover selective small-molecule inhibitors. Here, we report a discovery pipeline encompassing a biochemical screening campaign and a set of complementary biochemical, biophysical, and cell-based readouts for rigorous characterization of the screen output. The identified small molecules, TH6342 and analogs, accompanied by inactive control TH7126, demonstrated specific, low µM potency against both physiological and oncology-drug-derived substrates. By coupling kinetic studies with thermal shift assays, we reveal the inhibitory mechanism of TH6342 and analogs, which engage pre-tetrameric SAMHD1 and deter oligomerization and allosteric activation without occupying nucleotide-binding pockets. Altogether, our study diversifies inhibitory modes against SAMHD1, and the discovery pipeline reported herein represents a thorough framework for future SAMHD1 inhibitor development.

8.
Front Pharmacol ; 15: 1401599, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39050757

RESUMO

With over 450 genes, solute carriers (SLCs) constitute the largest transporter superfamily responsible for the uptake and efflux of nutrients, metabolites, and xenobiotics in human cells. SLCs are associated with a wide variety of human diseases, including cancer, diabetes, and metabolic and neurological disorders. They represent an important therapeutic target class that remains only partly exploited as therapeutics that target SLCs are scarce. Additionally, many small molecules reported in the literature to target SLCs are poorly characterized. Both features may be due to the difficulty of developing SLC transport assays that fulfill the quality criteria for high-throughput screening. Here, we report one of the main limitations hampering assay development within the RESOLUTE consortium: the lack of a resource providing high-quality information on SLC tool compounds. To address this, we provide a systematic annotation of tool compounds targeting SLCs. We first provide an overview on RESOLUTE assays. Next, we present a list of SLC-targeting compounds collected from the literature and public databases; we found that most data sources lacked specificity data. Finally, we report on experimental tests of 19 selected compounds against a panel of 13 SLCs from seven different families. Except for a few inhibitors, which were active on unrelated SLCs, the tested inhibitors demonstrated high selectivity for their reported targets. To make this knowledge easily accessible to the scientific community, we created an interactive dashboard displaying the collected data in the RESOLUTE web portal (https://re-solute.eu). We anticipate that our open-access resources on assays and compounds will support the development of future drug discovery campaigns for SLCs.

9.
ChemMedChem ; 17(18): e202200274, 2022 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-35712863

RESUMO

Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) is a mitochondrial 1-carbon metabolism enzyme, which is an attractive anticancer drug target as it is highly upregulated in cancer but is not expressed in healthy adult cells. Selective MTHFD2 inhibitors could therefore offer reduced side-effects during treatment, which are common with antifolate drugs that target other 1C-metabolism enzymes. This task is challenging however, as MTHFD2 shares high sequence identity with the constitutively expressed isozymes cytosolic MTHFD1 and mitochondrial MTHFD2L. In fact, one of the most potent MTHFD2 inhibitors reported to date, TH7299, is actually more active against MTHFD1 and MTHFD2L. While structures of MTHFD2 and MTHFD1 exist, no MTHFD2L structures are available. We determined the first structure of MTHFD2L and its complex with TH7299, which reveals the structural basis for its highly potent MTHFD2L inhibition. Detailed analysis of the MTHFD2L structure presented here clearly highlights the challenges associated with developing truly isoform-selective MTHFD2 inhibitors.


Assuntos
Antineoplásicos , Antagonistas do Ácido Fólico , Metilenotetra-Hidrofolato Desidrogenase (NADP)/química , Carbono , Humanos , Isoenzimas/metabolismo , Metilenotetra-Hidrofolato Desidrogenase (NADP)/metabolismo
10.
Science ; 376(6600): 1471-1476, 2022 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-35737787

RESUMO

Oxidative DNA damage is recognized by 8-oxoguanine (8-oxoG) DNA glycosylase 1 (OGG1), which excises 8-oxoG, leaving a substrate for apurinic endonuclease 1 (APE1) and initiating repair. Here, we describe a small molecule (TH10785) that interacts with the phenylalanine-319 and glycine-42 amino acids of OGG1, increases the enzyme activity 10-fold, and generates a previously undescribed ß,δ-lyase enzymatic function. TH10785 controls the catalytic activity mediated by a nitrogen base within its molecular structure. In cells, TH10785 increases OGG1 recruitment to and repair of oxidative DNA damage. This alters the repair process, which no longer requires APE1 but instead is dependent on polynucleotide kinase phosphatase (PNKP1) activity. The increased repair of oxidative DNA lesions with a small molecule may have therapeutic applications in various diseases and aging.


Assuntos
Dano ao DNA , DNA Glicosilases , Reparo do DNA , Estresse Oxidativo , Biocatálise/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , DNA Glicosilases/química , DNA Glicosilases/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Ativação Enzimática , Glicina/química , Humanos , Ligantes , Estresse Oxidativo/genética , Fenilalanina/química , Especificidade por Substrato
11.
Cell Chem Biol ; 28(12): 1693-1702.e6, 2021 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-34192523

RESUMO

Ganciclovir (GCV) is the first-line therapy against human cytomegalovirus (HCMV), a widespread infection that is particularly dangerous for immunodeficient individuals. Closely resembling deoxyguanosine triphosphate, the tri-phosphorylated metabolite of GCV (GCV-TP) is preferentially incorporated by the viral DNA polymerase, thereby terminating chain extension and, eventually, viral replication. However, the treatment outcome of GCV varies greatly among individuals, therefore warranting better understanding of its metabolism. Here we show that NUDT15, a Nudix hydrolase known to metabolize thiopurine triphosphates, can similarly hydrolyze GCV-TP through biochemical studies and co-crystallization of the NUDT15/GCV-TP complex. More critically, GCV efficacy was potentiated in HCMV-infected cells following NUDT15 depletion by RNAi or inhibition by an in-house-developed, nanomolar NUDT15 inhibitor, TH8321, suggesting that pharmacological targeting of NUDT15 is a possible avenue to improve existing anti-HCMV regimens. Collectively, the data further implicate NUDT15 as a broad-spectrum metabolic regulator of nucleoside analog therapeutics, such as thiopurines and GCV.


Assuntos
Antivirais/farmacologia , Citomegalovirus/efeitos dos fármacos , Ganciclovir/farmacologia , Pirofosfatases/metabolismo , Antivirais/química , Linhagem Celular Tumoral , Feminino , Ganciclovir/química , Humanos , Hidrólise , Testes de Sensibilidade Microbiana , Proteínas Recombinantes/metabolismo
12.
Front Chem ; 8: 443, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32548091

RESUMO

Computational chemistry has now been widely accepted as a useful tool for shortening lead times in early drug discovery. When selecting new potential drug targets, it is important to assess the likelihood of finding suitable starting points for lead generation before pursuing costly high-throughput screening campaigns. By exploiting available high-resolution crystal structures, an in silico druggability assessment can facilitate the decision of whether, and in cases where several protein family members exist, which of these to pursue experimentally. Many of the algorithms and software suites commonly applied for in silico druggability assessment are complex, technically challenging and not always user-friendly. Here we applied the intuitive open access servers of DoGSite, FTMap and CryptoSite to comprehensively predict ligand binding pockets, druggability scores and conformationally active regions of the NUDIX protein family. In parallel we analyzed potential ligand binding sites, their druggability and pocket parameter using Schrödinger's SiteMap. Then an in silico docking cascade of a subset of the ZINC FragNow library using the Glide docking program was performed to assess identified pockets for large-scale small-molecule binding. Subsequently, this initial dual ranking of druggable sites within the NUDIX protein family was benchmarked against experimental hit rates obtained both in-house and by others from traditional biochemical and fragment screening campaigns. The observed correlation suggests that the presented user-friendly workflow of a dual parallel in silico druggability assessment is applicable as a standalone method for decision on target prioritization and exclusion in future screening campaigns.

13.
ACS Omega ; 4(7): 11642-11656, 2019 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-31460271

RESUMO

Due to a polar or even charged binding interface, DNA-binding proteins are considered extraordinarily difficult targets for development of small-molecule ligands and only a handful of proteins have been targeted successfully to date. Recently, however, it has been shown that development of selective and efficient inhibitors of 8-oxoguanine DNA glycosylase is possible. Here, we describe the initial druggability assessment of DNA glycosylases in a computational setting and experimentally investigate several methods to target endonuclease VIII-like 1 (NEIL1) with small-molecule inhibitors. We find that DNA glycosylases exhibit good predicted druggability in both DNA-bound and -unbound states. Furthermore, we find catalytic sites to be highly flexible, allowing for a range of interactions and binding partners. One flexible catalytic site was rationalized for NEIL1 and further investigated experimentally using both a biochemical assay in the presence of DNA and a thermal shift assay in the absence of DNA.

14.
Science ; 362(6416): 834-839, 2018 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-30442810

RESUMO

The onset of inflammation is associated with reactive oxygen species and oxidative damage to macromolecules like 7,8-dihydro-8-oxoguanine (8-oxoG) in DNA. Because 8-oxoguanine DNA glycosylase 1 (OGG1) binds 8-oxoG and because Ogg1-deficient mice are resistant to acute and systemic inflammation, we hypothesized that OGG1 inhibition may represent a strategy for the prevention and treatment of inflammation. We developed TH5487, a selective active-site inhibitor of OGG1, which hampers OGG1 binding to and repair of 8-oxoG and which is well tolerated by mice. TH5487 prevents tumor necrosis factor-α-induced OGG1-DNA interactions at guanine-rich promoters of proinflammatory genes. This, in turn, decreases DNA occupancy of nuclear factor κB and proinflammatory gene expression, resulting in decreased immune cell recruitment to mouse lungs. Thus, we present a proof of concept that targeting oxidative DNA repair can alleviate inflammatory conditions in vivo.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Benzimidazóis/farmacologia , DNA Glicosilases/antagonistas & inibidores , Inibidores Enzimáticos/uso terapêutico , Expressão Gênica/efeitos dos fármacos , Inflamação/tratamento farmacológico , Piperidinas/farmacologia , Animais , Anti-Inflamatórios não Esteroides/uso terapêutico , Benzimidazóis/uso terapêutico , DNA Glicosilases/metabolismo , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/genética , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Técnicas de Inativação de Genes , Guanina/análogos & derivados , Guanina/antagonistas & inibidores , Guanina/metabolismo , Células HEK293 , Humanos , Inflamação/genética , Células Jurkat , Camundongos , Camundongos Mutantes , NF-kappa B/genética , NF-kappa B/metabolismo , Piperidinas/uso terapêutico , Regiões Promotoras Genéticas , Fator de Necrose Tumoral alfa/farmacologia
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