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1.
Biochemistry ; 2024 Aug 22.
Artículo en Inglés | MEDLINE | ID: mdl-39172504

RESUMEN

The identification of chemical starting points for the development of molecular glues is challenging. Here, we employed fragment screening and identified an allosteric stabilizer of the complex between 14-3-3 and a TAZ-derived peptide. The fragment binds preferentially to the 14-3-3/TAZ peptide complex and shows moderate stabilization in differential scanning fluorimetry and microscale thermophoresis. The binding site of the fragment was predicted by molecular dynamics calculations to be distant from the 14-3-3/TAZ peptide interface, located between helices 8 and 9 of the 14-3-3 protein. This site was confirmed by nuclear magnetic resonance and X-ray protein crystallography, revealing the first example of an allosteric stabilizer for 14-3-3 protein-protein interactions.

2.
J Biol Chem ; 298(12): 102629, 2022 12.
Artículo en Inglés | MEDLINE | ID: mdl-36273589

RESUMEN

mTORC1 and GCN2 are serine/threonine kinases that control how cells adapt to amino acid availability. mTORC1 responds to amino acids to promote translation and cell growth while GCN2 senses limiting amino acids to hinder translation via eIF2α phosphorylation. GCN2 is an appealing target for cancer therapies because malignant cells can harness the GCN2 pathway to temper the rate of translation during rapid amino acid consumption. To isolate new GCN2 inhibitors, we created cell-based, amino acid limitation reporters via genetic manipulation of Ddit3 (encoding the transcription factor CHOP). CHOP is strongly induced by limiting amino acids and in this context, GCN2-dependent. Using leucine starvation as a model for essential amino acid sensing, we unexpectedly discovered ATP-competitive PI3 kinase-related kinase inhibitors, including ATR and mTOR inhibitors like torins, completely reversed GCN2 activation in a time-dependent way. Mechanistically, via inhibiting mTORC1-dependent translation, torins increased intracellular leucine, which was sufficient to reverse GCN2 activation and the downstream integrated stress response including stress-induced transcriptional factor ATF4 expression. Strikingly, we found that general translation inhibitors mirrored the effects of torins. Therefore, we propose that mTOR kinase inhibitors concurrently inhibit different branches of amino acid sensing by a dual mechanism involving direct inhibition of mTOR and indirect suppression of GCN2 that are connected by effects on the translation machinery. Collectively, our results highlight distinct ways of regulating GCN2 activity.


Asunto(s)
Aminoácidos , Proteínas Serina-Treonina Quinasas , Transducción de Señal , Aminoácidos/genética , Aminoácidos/metabolismo , Factor 2 Eucariótico de Iniciación/genética , Factor 2 Eucariótico de Iniciación/metabolismo , Leucina/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Fosforilación , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Humanos , Animales , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo
3.
Int J Mol Sci ; 22(2)2021 Jan 08.
Artículo en Inglés | MEDLINE | ID: mdl-33430060

RESUMEN

Human cytomegalovirus (HCMV) is a human pathogenic herpesvirus associated with a variety of clinical symptoms. Current antiviral therapy is not always effective, so that improved drug classes and drug-targeting strategies are needed. Particularly host-directed antivirals, including pharmaceutical kinase inhibitors (PKIs), may help to overcome problems of drug resistance. Here, we focused on utilizing a selection of clinically relevant PKIs and determined their anticytomegaloviral efficacies. Particularly, PKIs directed to host or viral cyclin-dependent kinases, i.e., abemaciclib, LDC4297 and maribavir, exerted promising profiles against human and murine cytomegaloviruses. The anti-HCMV in vitro activity of the approved anti-cancer drug abemaciclib was confirmed in vivo using our luciferase-based murine cytomegalovirus (MCMV) animal model in immunocompetent mice. To assess drug combinations, we applied the Bliss independence checkerboard and Loewe additivity fixed-dose assays in parallel. Results revealed that (i) both affirmative approaches provided valuable information on anti-CMV drug efficacies and interactions, (ii) the analyzed combinations comprised additive, synergistic or antagonistic drug interactions consistent with the drugs' antiviral mode-of-action, (iii) the selected PKIs, especially LDC4297, showed promising inhibitory profiles, not only against HCMV but also other α-, ß- and γ-herpesviruses, and specifically, (iv) the combination treatment with LDC4297 and maribavir revealed a strong synergism against HCMV, which might open doors towards novel clinical options in the near future. Taken together, this study highlights the potential of therapeutic drug combinations of current developmental/preclinical PKIs.


Asunto(s)
Infecciones por Citomegalovirus/tratamiento farmacológico , Farmacorresistencia Viral/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Replicación Viral/genética , Aminopiridinas/farmacología , Animales , Antivirales/farmacología , Bencimidazoles/farmacología , Línea Celular , Citomegalovirus/efectos de los fármacos , Citomegalovirus/patogenicidad , Infecciones por Citomegalovirus/genética , Infecciones por Citomegalovirus/virología , Combinación de Medicamentos , Ganciclovir/farmacología , Humanos , Ratones , Pirazoles/farmacología , Ribonucleósidos/farmacología , Triazinas/farmacología , Replicación Viral/efectos de los fármacos
4.
Nature ; 507(7493): 508-12, 2014 Mar 27.
Artículo en Inglés | MEDLINE | ID: mdl-24553136

RESUMEN

Tumour metastasis is the primary cause of mortality in cancer patients and remains the key challenge for cancer therapy. New therapeutic approaches to block inhibitory pathways of the immune system have renewed hopes for the utility of such therapies. Here we show that genetic deletion of the E3 ubiquitin ligase Cbl-b (casitas B-lineage lymphoma-b) or targeted inactivation of its E3 ligase activity licenses natural killer (NK) cells to spontaneously reject metastatic tumours. The TAM tyrosine kinase receptors Tyro3, Axl and Mer (also known as Mertk) were identified as ubiquitylation substrates for Cbl-b. Treatment of wild-type NK cells with a newly developed small molecule TAM kinase inhibitor conferred therapeutic potential, efficiently enhancing anti-metastatic NK cell activity in vivo. Oral or intraperitoneal administration using this TAM inhibitor markedly reduced murine mammary cancer and melanoma metastases dependent on NK cells. We further report that the anticoagulant warfarin exerts anti-metastatic activity in mice via Cbl-b/TAM receptors in NK cells, providing a molecular explanation for a 50-year-old puzzle in cancer biology. This novel TAM/Cbl-b inhibitory pathway shows that it might be possible to develop a 'pill' that awakens the innate immune system to kill cancer metastases.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Células Asesinas Naturales/inmunología , Neoplasias Mamarias Experimentales/patología , Melanoma Experimental/patología , Metástasis de la Neoplasia/inmunología , Proteínas Proto-Oncogénicas c-cbl/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Anticoagulantes/farmacología , Anticoagulantes/uso terapéutico , Femenino , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/metabolismo , Masculino , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/inmunología , Melanoma Experimental/tratamiento farmacológico , Melanoma Experimental/genética , Melanoma Experimental/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Metástasis de la Neoplasia/tratamiento farmacológico , Metástasis de la Neoplasia/prevención & control , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-cbl/deficiencia , Proteínas Proto-Oncogénicas c-cbl/genética , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/deficiencia , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación , Warfarina/farmacología , Warfarina/uso terapéutico , Tirosina Quinasa c-Mer , Tirosina Quinasa del Receptor Axl
5.
Angew Chem Int Ed Engl ; 58(46): 16617-16628, 2019 11 11.
Artículo en Inglés | MEDLINE | ID: mdl-31454140

RESUMEN

The Hedgehog (Hh) signaling pathway is crucial for vertebrate embryonic development, tissue homeostasis and regeneration. Hh signaling is upregulated in basal cell carcinoma and medulloblastoma and Hh pathway inhibitors targeting the Smoothened (SMO) protein are in clinical use. However, the signaling cascade is incompletely understood and novel druggable proteins in the pathway are in high demand. We describe the discovery of the Hh-pathway modulator Pipinib by means of cell-based screening. Target identification and validation revealed that Pipinib selectively inhibits phosphatidylinositol 4-kinase IIIß (PI4KB) and suppresses GLI-mediated transcription and Hh target gene expression by impairing SMO translocation to the cilium. Therefore, inhibition of PI4KB and, consequently, reduction in phosphatidyl-4-phosphate levels may be considered an alternative approach to inhibit SMO function and thus, Hedgehog signaling.


Asunto(s)
Antineoplásicos/farmacología , Proteínas Hedgehog/antagonistas & inhibidores , Antígenos de Histocompatibilidad Menor/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Transducción de Señal/efectos de los fármacos , Tiofenos/farmacología , Animales , Antineoplásicos/química , Línea Celular , Supervivencia Celular/efectos de los fármacos , Cilios/metabolismo , Expresión Génica/efectos de los fármacos , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Humanos , Ratones , Antígenos de Histocompatibilidad Menor/genética , Morfolinas/farmacología , Osteogénesis/efectos de los fármacos , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Purinas/farmacología , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Relación Estructura-Actividad , Tiofenos/química
6.
Antimicrob Agents Chemother ; 59(4): 2062-71, 2015 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-25624324

RESUMEN

Protein kinases represent central and multifunctional regulators of a balanced virus-host interaction. Cyclin-dependent protein kinase 7 (CDK7) plays crucial regulatory roles in cell cycle and transcription, both connected with the replication of many viruses. Previously, we developed a CDK7 inhibitor, LDC4297, that inhibits CDK7 in vitro in the nano-picomolar range. Novel data from a kinome-wide evaluation (>330 kinases profiled in vitro) demonstrate a kinase selectivity. Importantly, we provide first evidence for the antiviral potential of the CDK7 inhibitor LDC4297, i.e., in exerting a block of the replication of human cytomegalovirus (HCMV) in primary human fibroblasts at nanomolar concentrations (50% effective concentration, 24.5 ± 1.3 nM). As a unique feature compared to approved antiherpesviral drugs, inhibition occurred already at the immediate-early level of HCMV gene expression. The mode of antiviral action was considered multifaceted since CDK7-regulated cellular factors that are supportive of HCMV replication were substantially affected by the inhibitors. An effect of LDC4297 was identified in the interference with HCMV-driven inactivation of retinoblastoma protein (Rb), a regulatory step generally considered a hallmark of herpesviral replication. In line with this finding, a broad inhibitory activity of the drug could be demonstrated against a selection of human and animal herpesviruses and adenoviruses, whereas other viruses only showed intermediate drug sensitivity. Summarized, the CDK7 inhibitor LDC4297 is a promising candidate for further antiviral drug development, possibly offering new options for a comprehensive approach to antiviral therapy.


Asunto(s)
Antivirales/farmacología , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Pirazoles/farmacología , Triazinas/farmacología , Adenoviridae/efectos de los fármacos , Animales , Supervivencia Celular/efectos de los fármacos , Citomegalovirus/efectos de los fármacos , Fibroblastos/virología , Regulación Viral de la Expresión Génica/efectos de los fármacos , Herpesviridae/efectos de los fármacos , Humanos , Ratones , Fosforilación , Replicación Viral/efectos de los fármacos
7.
Eur J Med Chem ; 265: 116053, 2024 Feb 05.
Artículo en Inglés | MEDLINE | ID: mdl-38141285

RESUMEN

The colony-stimulating factor 1 receptor (CSF1R) is an attractive target for inflammation disorders and cancers. Based on a series of pyrrolo[2,3-d]pyrimidine containing two carbo-aromatic rings, we have searched for new CSF1R inhibitors having a higher fraction of sp3-atoms. The phenyl unit in the 4-amino group could efficiently be replaced by tetrahydropyran (THP) retaining inhibitor potency. Exchanging the 6-aryl group with cyclohex-2-ene units also resulted in highly potent compounds, while fully saturated ring systems at C-6 led to a loss of activity. The structure-activity relationship study evaluating THP containing pyrrolo[2,3-d]pyrimidine derivates identified several highly active inhibitors by enzymatic studies. A comparison of 11 pairs of THP and aromatic compounds showed that inhibitors containing THP had clear benefits in terms of enzymatic potency, solubility, and cell toxicity. Guided by cellular experiments in Ba/F3 cells, five CSF1R inhibitors were further profiled in ADME assays, indicating the para-aniline derivative 16t as the most attractive compound for further development.


Asunto(s)
Pirimidinas , Proteínas Tirosina Quinasas Receptoras , Pirimidinas/farmacología , Pirroles/farmacología , Relación Estructura-Actividad , Inhibidores de Proteínas Quinasas/farmacología
8.
Pharmaceutics ; 16(2)2024 Jan 23.
Artículo en Inglés | MEDLINE | ID: mdl-38399219

RESUMEN

The repertoire of currently available antiviral drugs spans therapeutic applications against a number of important human pathogens distributed worldwide. These include cases of the pandemic severe acute respiratory coronavirus type 2 (SARS-CoV-2 or COVID-19), human immunodeficiency virus type 1 (HIV-1 or AIDS), and the pregnancy- and posttransplant-relevant human cytomegalovirus (HCMV). In almost all cases, approved therapies are based on direct-acting antivirals (DAAs), but their benefit, particularly in long-term applications, is often limited by the induction of viral drug resistance or side effects. These issues might be addressed by the additional use of host-directed antivirals (HDAs). As a strong input from long-term experiences with cancer therapies, host protein kinases may serve as HDA targets of mechanistically new antiviral drugs. The study demonstrates such a novel antiviral strategy by targeting the major virus-supportive host kinase CDK7. Importantly, this strategy focuses on highly selective, 3D structure-derived CDK7 inhibitors carrying a warhead moiety that mediates covalent target binding. In summary, the main experimental findings of this study are as follows: (1) the in vitro verification of CDK7 inhibition and selectivity that confirms the warhead covalent-binding principle (by CDK-specific kinase assays), (2) the highly pronounced antiviral efficacies of the hit compounds (in cultured cell-based infection models) with half-maximal effective concentrations that reach down to picomolar levels, (3) a particularly strong potency of compounds against strains and reporter-expressing recombinants of HCMV (using infection assays in primary human fibroblasts), (4) additional activity against further herpesviruses such as animal CMVs and VZV, (5) unique mechanistic properties that include an immediate block of HCMV replication directed early (determined by Western blot detection of viral marker proteins), (6) a substantial drug synergism in combination with MBV (measured by a Loewe additivity fixed-dose assay), and (7) a strong sensitivity of clinically relevant HCMV mutants carrying MBV or ganciclovir resistance markers. Combined, the data highlight the huge developmental potential of this host-directed antiviral targeting concept utilizing covalently binding CDK7 inhibitors.

9.
Antibiotics (Basel) ; 12(7)2023 Jun 28.
Artículo en Inglés | MEDLINE | ID: mdl-37508212

RESUMEN

Bacteria of the genus Streptomyces produce various specialized metabolites. Single biosynthetic gene clusters (BGCs) can give rise to different products that can vary in terms of their biological activities. For example, for alnumycin and the shunt product K115, antimicrobial activity was described, while no antimicrobial activity was detected for the shunt product 1,6-dihydro 8-propylanthraquinone. To investigate the antibacterial activity of 1,6-dihydro 8-propylanthraquinone, we produced alnumycin and 1,6-dihydro 8-propylanthraquinone from a Streptomyces isolate containing the alnumycin BGC. The strain was cultivated in liquid glycerol-nitrate-casein medium (GN), and both compounds were isolated using an activity and mass spectrometry-guided purification. The structures were validated via nuclear magnetic resonance (NMR) spectroscopy. A minimal inhibitory concentration (MIC) test revealed that 1,6-dihydro 8-propylanthraquinone exhibits antimicrobial activity against E. coli ΔtolC, B. subtilis, an S. aureus type strain, and a vancomycin intermediate-resistance S. aureus strain (VISA). Activity of 1,6-dihydro 8-propylanthraquinone against E. coli ΔtolC was approximately 10-fold higher than that of alnumycin. We were unable to confirm gyrase inhibition for either compound and believe that the modes of action of both compounds are worth reinvestigating.

10.
Cells ; 12(8)2023 04 14.
Artículo en Inglés | MEDLINE | ID: mdl-37190072

RESUMEN

Herpesviral nuclear egress is a regulated process of viral capsid nucleocytoplasmic release. Due to the large capsid size, a regular transport via the nuclear pores is unfeasible, so that a multistage-regulated export pathway through the nuclear lamina and both leaflets of the nuclear membrane has evolved. This process involves regulatory proteins, which support the local distortion of the nuclear envelope. For human cytomegalovirus (HCMV), the nuclear egress complex (NEC) is determined by the pUL50-pUL53 core that initiates multicomponent assembly with NEC-associated proteins and capsids. The transmembrane NEC protein pUL50 serves as a multi-interacting determinant that recruits regulatory proteins by direct and indirect contacts. The nucleoplasmic core NEC component pUL53 is strictly associated with pUL50 in a structurally defined hook-into-groove complex and is considered as the potential capsid-binding factor. Recently, we validated the concept of blocking the pUL50-pUL53 interaction by small molecules as well as cell-penetrating peptides or an overexpression of hook-like constructs, which can lead to a pronounced degree of antiviral activity. In this study, we extended this strategy by utilizing covalently binding warhead compounds, originally designed as binders of distinct cysteine residues in target proteins, such as regulatory kinases. Here, we addressed the possibility that warheads may likewise target viral NEC proteins, building on our previous crystallization-based structural analyses that revealed distinct cysteine residues in positions exposed from the hook-into-groove binding surface. To this end, the antiviral and NEC-binding properties of a selection of 21 warhead compounds were investigated. The combined findings are as follows: (i) warhead compounds exhibited a pronounced anti-HCMV potential in cell-culture-based infection models; (ii) computational analysis of NEC primary sequences and 3D structures revealed cysteine residues exposed to the hook-into-groove interaction surface; (iii) several of the active hit compounds exhibited NEC-blocking activity, as shown at the single-cell level by confocal imaging; (iv) the clinically approved warhead drug ibrutinib exerted a strong inhibitory impact on the pUL50-pUL53 core NEC interaction, as demonstrated by the NanoBiT assay system; and (v) the generation of recombinant HCMV ∆UL50-ΣUL53, allowing the assessment of viral replication under conditional expression of the viral core NEC proteins, was used for characterizing viral replication and a mechanistic evaluation of ibrutinib antiviral efficacy. Combined, the results point to a rate-limiting importance of the HCMV core NEC for viral replication and to the option of exploiting this determinant by the targeting of covalently NEC-binding warhead compounds.


Asunto(s)
Antivirales , Citomegalovirus , Humanos , Antivirales/farmacología , Antivirales/metabolismo , Cisteína/metabolismo , Membrana Nuclear/metabolismo , Núcleo Celular/metabolismo , Proteínas Virales/metabolismo
11.
Pharmaceutics ; 15(12)2023 Nov 27.
Artículo en Inglés | MEDLINE | ID: mdl-38140021

RESUMEN

Despite the availability of currently approved antiviral drugs, infections with human cytomegalovirus (HCMV) still cause clinically challenging, sometimes life-threatening situations. There is an urgent need for enhanced anti-HCMV drugs that offer improved efficacy, reduced dosages and options for long-term treatment without risk of the development of viral drug resistance. Recently, we reported the pronounced anti-HCMV efficacy of pharmacological inhibitors of cyclin-dependent kinases (CDKs), in particular, the potential of utilizing drug synergies upon combination treatment with inhibitors of host CDKs and the viral CDK-like kinase pUL97 (vCDK/pUL97). Here, we expand this finding by further assessing the in vitro synergistic antiviral interaction between vCDK and CDK inhibitors towards HCMV as well as non-human cytomegaloviruses. An extension of this synergy approach was achieved in vivo by using the recombinant MCMV-UL97/mouse model, confirming the high potential of combination treatment with the clinically approved vCDK inhibitor maribavir (MBV) and the developmental CDK7 inhibitor LDC4297. Moreover, mechanistic aspects of this synergistic drug combination were illustrated on the levels of intracellular viral protein transport and viral genome replication. The analysis of viral drug resistance did not reveal resistance formation in the case of MBV + LDC4297 combination treatment. Spanning various investigational levels, these new results strongly support our concept, employing the great potential of anti-HCMV synergistic drug treatment.

12.
Virus Res ; 335: 199200, 2023 10 02.
Artículo en Inglés | MEDLINE | ID: mdl-37591314

RESUMEN

Human cytomegalovirus (HCMV) infection is shaped by a tightly regulated interplay between viral and cellular proteins. Distinct kinase activities, such as the viral cyclin-dependent kinase ortholog (vCDK) pUL97 and cellular CDK7 are both crucial for efficient viral replication. Previously, we reported that both kinases, vCDK/pUL97 and CDK7, interact with cyclin H, thereby achieving an enhanced level of kinase activity and overall functionality in viral replication. Here we provide a variety of novel results, as generated on a methodologically extended basis, and present a concept for the codetermination of viral replication efficiency through these kinase activities: (i) cyclin H expression, in various human cell types, is substantially upregulated by strains of HCMV including the clinically relevant HCMV Merlin; (ii) vCDK/pUL97 interacts with human cyclin H in both HCMV-infected and plasmid-transfected cell systems; (iii) a doxycycline-inducible shRNA-dependent knock-down (KD) of cyclin H significantly reduces pUL97 activity (qSox in vitro kinase assay); (iv) accordingly, pUL97 in vitro kinase activity is seen significantly increased upon addition of recombinant cyclin H; (v) as a point of specific importance, human CDK7 activity shows an increase by vCDK/pUL97-mediated trans-stimulation (whereas pUL97 is not stimulated by CDK7); (vi) phosphosite-specific antibodies indicate an upregulated CDK7 phosphorylation upon HCMV infection, as mediated through a pUL97-specific modulatory effect (i.e. shown by pUL97 inhibitor treatment or pUL97-deficient viral mutant); (vii) finally, an efficient KD of cyclin H in primary fibroblasts generally results in an impaired HCMV replication efficiency as measured on protein and genomic levels. These results show evidence for the codetermination of viral replication by vCDK/pUL97, cyclin H and CDK7, thus supporting the specific importance of cyclin H as a central regulatory factor, and suggesting novel targeting options for antiviral drugs.


Asunto(s)
Quinasas Ciclina-Dependientes , Citomegalovirus , Humanos , Antivirales , Ciclina H , Quinasas Ciclina-Dependientes/genética , Citomegalovirus/genética , Fosforilación
13.
J Med Chem ; 66(10): 6959-6980, 2023 05 25.
Artículo en Inglés | MEDLINE | ID: mdl-37191268

RESUMEN

Colony-stimulating factor-1 receptor (CSF1R) is a receptor tyrosine kinase that controls the differentiation and maintenance of most tissue-resident macrophages, and the inhibition of CSF1R has been suggested as a possible therapy for a range of human disorders. Herein, we present the synthesis, development, and structure-activity relationship of a series of highly selective pyrrolo[2,3-d]pyrimidines, showing subnanomolar enzymatic inhibition of this receptor and with excellent selectivity toward other kinases in the platelet-derived growth factor receptor (PDGFR) family. The crystal structure of the protein and 23 revealed that the binding conformation of the protein is DFG-out-like. The most promising compounds in this series were profiled for cellular potency and subjected to pharmacokinetic profiling and in vivo stability, indicating that this compound class could be relevant in a potential disease setting. Additionally, these compounds inhibited primarily the autoinhibited form of the receptor, contrasting the behavior of pexidartinib, which could explain the exquisite selectivity of these structures.


Asunto(s)
Pirimidinas , Proteínas Tirosina Quinasas Receptoras , Humanos , Relación Estructura-Actividad , Pirimidinas/química , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/química
14.
Cell Chem Biol ; 30(6): 573-590.e6, 2023 06 15.
Artículo en Inglés | MEDLINE | ID: mdl-37130519

RESUMEN

The natural product family of the fusicoccanes (FCs) has been shown to display anti-cancer activity, especially when combined with established therapeutic agents. FCs stabilize 14-3-3 protein-protein interactions (PPIs). Here, we tested combinations of a small library of FCs with interferon α (IFNα) on different cancer cell lines and report a proteomics approach to identify the specific 14-3-3 PPIs that are induced by IFNα and stabilized by FCs in OVCAR-3 cells. Among the identified 14-3-3 target proteins are THEMIS2, receptor interacting protein kinase 2 (RIPK2), EIF2AK2, and several members of the LDB1 complex. Biophysical and structural biology studies confirm these 14-3-3 PPIs as physical targets of FC stabilization, and transcriptome as well as pathway analyses suggest possible explanations for the observed synergistic effect of IFNα/FC treatment on cancer cells. This study elucidates the polypharmacological effects of FCs in cancer cells and identifies potential targets from the vast interactome of 14-3-3s for therapeutic intervention in oncology.


Asunto(s)
Interferón-alfa , Neoplasias Ováricas , Humanos , Femenino , Interferón-alfa/farmacología , Apoptosis , Línea Celular Tumoral , Muerte Celular
15.
Eur J Med Chem ; 255: 115344, 2023 Jul 05.
Artículo en Inglés | MEDLINE | ID: mdl-37141705

RESUMEN

The colony-stimulating factor 1 receptor (CSF1R) plays an important role in the regulation of many inflammatory processes, and overexpression of the kinase is implicated in several disease states. Identifying selective, small-molecule inhibitors of CSF1R may be a crucial step toward treating these disorders. Through modelling, synthesis, and a systematic structure-activity relationship study, we have identified a number of potent and highly selective purine-based inhibitors of CSF1R. The optimized 6,8-disubstituted antagonist, compound 9, has enzymatic IC50 of 0.2 nM, and displays a strong affinity toward the autoinhibited form of CSF1R, contrasting that of other previously reported inhibitors. As a result of its binding mode, the inhibitor shows excellent selectivity (Selectivity score: 0.06), evidenced by profiling towards a panel of 468 kinases. In cell-based assays, this inhibitor shows dose-dependent blockade of CSF1-mediated downstream signalling in murine bone marrow-derived macrophages (IC50 = 106 nM) as well as disruption of osteoclast differentiation at nanomolar levels. In vivo experiments, however, indicate that improve metabolic stability is needed in order to further progress this compound class.


Asunto(s)
Macrófagos , Osteoclastos , Animales , Ratones , Proteínas Tirosina Quinasas Receptoras , Diferenciación Celular , Purinas/farmacología , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos
16.
Nat Commun ; 14(1): 8103, 2023 Dec 11.
Artículo en Inglés | MEDLINE | ID: mdl-38081825

RESUMEN

Autonomous migration is essential for the function of immune cells such as neutrophils and plays an important role in numerous diseases. The ability to routinely measure or target it would offer a wealth of clinical applications. Video microscopy of live cells is ideal for migration analysis, but cannot be performed at sufficiently high-throughput (HT). Here we introduce ComplexEye, an array microscope with 16 independent aberration-corrected glass lenses spaced at the pitch of a 96-well plate to produce high-resolution movies of migrating cells. With the system, we enable HT migration analysis of immune cells in 96- and 384-well plates with very energy-efficient performance. We demonstrate that the system can measure multiple clinical samples simultaneously. Furthermore, we screen 1000 compounds and identify 17 modifiers of migration in human neutrophils in just 4 days, a task that requires 60-times longer with a conventional video microscope. ComplexEye thus opens the field of phenotypic HT migration screens and enables routine migration analysis for the clinical setting.


Asunto(s)
Cristalino , Lentes , Humanos , Microscopía , Microscopía por Video , Movimiento Celular
17.
Commun Biol ; 5(1): 1206, 2022 11 09.
Artículo en Inglés | MEDLINE | ID: mdl-36352263

RESUMEN

Analysis of agonist-driven phosphorylation of G protein-coupled receptors (GPCRs) can provide valuable insights into the receptor activation state and ligand pharmacology. However, to date, assessment of GPCR phosphorylation using high-throughput applications has been challenging. We have developed and validated a bead-based immunoassay for the quantitative assessment of agonist-induced GPCR phosphorylation that can be performed entirely in multiwell cell culture plates. The assay involves immunoprecipitation of affinity-tagged receptors using magnetic beads followed by protein detection using phosphorylation state-specific and phosphorylation state-independent anti-GPCR antibodies. As proof of concept, five prototypical GPCRs (MOP, C5a1, D1, SST2, CB2) were treated with different agonizts and antagonists, and concentration-response curves were generated. We then extended our approach to establish selective cellular GPCR kinase (GRK) inhibitor assays, which led to the rapid identification of a selective GRK5/6 inhibitor (LDC8988) and a highly potent pan-GRK inhibitor (LDC9728). In conclusion, this versatile GPCR phosphorylation assay can be used extensively for ligand profiling and inhibitor screening.


Asunto(s)
Receptores Acoplados a Proteínas G , Fosforilación , Ligandos , Receptores Acoplados a Proteínas G/metabolismo , Inmunoensayo
18.
J Gen Virol ; 92(Pt 7): 1519-1531, 2011 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-21450947

RESUMEN

Cyclin-dependent protein kinases (CDKs) are important regulators of cellular processes and are functionally integrated into the replication of human cytomegalovirus (HCMV). Recently, a regulatory impact of CDK activity on the viral mRNA export factor pUL69 was shown. Here, specific aspects of the mode of interaction between CDK9/cyclin T1 and pUL69 are described. Intracellular localization was studied in the presence of a novel selective CDK9 inhibitor, R22, which exerts anti-cytomegaloviral activity in vitro. A pronounced R22-induced formation of nuclear speckled aggregation of pUL69 was demonstrated. Multi-labelling confocal laser-scanning microscopy revealed that CDK9 and cyclin T1 co-localized perfectly with pUL69 in individual speckles. The effects were similar to those described recently for the broad CDK inhibitor roscovitine. Co-immunoprecipitation and yeast two-hybrid analyses showed that cyclin T1 interacted with both CDK9 and pUL69. The interaction region of pUL69 for cyclin T1 could be attributed to aa 269-487. Moreover, another component of CDK inhibitor-induced speckled aggregates was identified with RNA polymerase II, supporting earlier reports that strongly suggested an association of pUL69 with transcription complexes. Interestingly, when using a UL69-deleted recombinant HCMV, no speckled aggregates were formed by CDK inhibitor treatment. This indicated that pUL69 is the defining component of aggregates and generally may represent a crucial viral interactor of cyclin T1. In conclusion, these data emphasize that HCMV inter-regulation with CDK9/cyclin T1 is at least partly based on a pUL69-cylin T1 interaction, thus contributing to the importance of CDK9 for HCMV replication.


Asunto(s)
Núcleo Celular/enzimología , Ciclina T/metabolismo , Quinasa 9 Dependiente de la Ciclina/metabolismo , Infecciones por Citomegalovirus/metabolismo , Citomegalovirus/fisiología , Transactivadores/metabolismo , Replicación Viral , Línea Celular , Núcleo Celular/genética , Núcleo Celular/virología , Ciclina T/genética , Quinasa 9 Dependiente de la Ciclina/genética , Citomegalovirus/genética , Infecciones por Citomegalovirus/enzimología , Infecciones por Citomegalovirus/virología , Humanos , Unión Proteica , Transporte de Proteínas , Transactivadores/genética
19.
Antiviral Res ; 161: 63-69, 2019 01.
Artículo en Inglés | MEDLINE | ID: mdl-30452929

RESUMEN

Infections with the human cytomegalovirus (HCMV) cause serious medical problems including organ rejection and congenital infection. Treatment of HCMV infections with currently available medication targeting viral enzymes is often accompanied with severe side effects and the occurrence of drug-resistant viruses. This demands novel therapeutical approaches like targeting genetically stable host cell proteins that are crucial for virus replication. Although numerous experimental drugs with promising in vitro efficacy have been identified, the lack of available data in animal models limits their potential for further clinical development. Recently, we described the very strong in vitro antiherpesviral activity of the NF-κB inhibitor TF27 and the CDK7 inhibitor LDC4297 at low nanomolar concentrations. In the present study, we present first data for the in vivo efficacy of both experimental drugs using an established cytomegalovirus animal model (murine CMV replication in immunodefective Rag -/- mice). The main findings of this study are (i) a strong inhibitory potency against beta- and gamma-herpesviruses of both compounds in vitro, (ii) even more important, a pronounced anticytomegaloviral activity also exerted in vivo, that resulted from (iii) a restriction of viral replication to the site of infection, thus preventing organ dissemination, (iv) in the absence of major compound-associated adverse events. Thus, we provide evidence for a strong antiviral potency in vivo and proof-of-concept for both drugs, which may encourage their further drug development, possibly including pharmacologically optimized derivatives, for a potential use in future antiherpesviral treatment.


Asunto(s)
Antivirales/farmacología , Infecciones por Citomegalovirus/tratamiento farmacológico , Muromegalovirus/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Animales , Modelos Animales de Enfermedad , Drogas en Investigación/farmacología , Ratones , Ratones Noqueados , Pruebas de Sensibilidad Microbiana , Muromegalovirus/fisiología , Prueba de Estudio Conceptual , Pirazoles/farmacología , Triazinas/farmacología
20.
J Med Chem ; 62(22): 10167-10181, 2019 11 27.
Artículo en Inglés | MEDLINE | ID: mdl-31647655

RESUMEN

In this work, we demonstrate that the indole-oxazole-pyrrole framework of the breitfussin family of natural products is a promising scaffold for kinase inhibition. Six new halogenated natural products, breitfussin C-H (3 - 8) were isolated and characterized from the Arctic, marine hydrozoan Thuiaria breitfussi. The structures of two of the new natural products were also confirmed by total synthesis. Two of the breitfussins (3 and 4) were found to selectively inhibit the survival of several cancer cell lines, with the lowest IC50 value of 340 nM measured against the drug-resistant triple negative breast cancer cell line MDA-MB-468, while leaving the majority of the tested cell lines not or significantly less affected. When tested against panels of protein kinases, 3 gave IC50 and Kd values as low as 200 and 390 nM against the PIM1 and DRAK1 kinases, respectively. The activity was confirmed to be mediated through ATP competitive binding in the ATP binding pocket of the kinases. Furthermore, evaluation of potential off-target and toxicological effects, as well as relevant in vitro ADME parameters for 3 revealed that the breitfussin scaffold holds promise for the development of selective kinase inhibitors.


Asunto(s)
Antineoplásicos/farmacología , Productos Biológicos/química , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Adenosina Trifosfato/metabolismo , Animales , Antineoplásicos/química , Regiones Árticas , Sitios de Unión , Productos Biológicos/aislamiento & purificación , Productos Biológicos/farmacología , Línea Celular Tumoral , Embrión no Mamífero/efectos de los fármacos , Femenino , Humanos , Hidrocarburos Bromados/química , Hidrozoos/química , Indoles/química , Ratones , Simulación del Acoplamiento Molecular , Estructura Molecular , Inhibidores de Proteínas Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/toxicidad , Proteínas Proto-Oncogénicas c-pim-1/química , Proteínas Proto-Oncogénicas c-pim-1/metabolismo , Pruebas de Toxicidad , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/patología , Pez Cebra/embriología
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