RESUMO
Vaccinia-related kinase 1 (VRK1) and the δ and ε isoforms of casein kinase 1 (CK1) are linked to various disease-relevant pathways. However, the lack of tool compounds for these kinases has significantly hampered our understanding of their cellular functions and therapeutic potential. Here, we describe the structure-based development of potent inhibitors of VRK1, a kinase highly expressed in various tumor types and crucial for cell proliferation and genome integrity. Kinome-wide profiling revealed that our compounds also inhibit CK1δ and CK1ε. We demonstrate that dihydropteridinones 35 and 36 mimic the cellular outcomes of VRK1 depletion. Complementary studies with existing CK1δ and CK1ε inhibitors suggest that these kinases may play overlapping roles in cell proliferation and genome instability. Together, our findings highlight the potential of VRK1 inhibition in treating p53-deficient tumors and possibly enhancing the efficacy of existing cancer therapies that target DNA stability or cell division.
Assuntos
Inibidores de Proteínas Quinases , Proteínas Serina-Treonina Quinases , Pteridinas , Humanos , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/síntese química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Pteridinas/farmacologia , Pteridinas/química , Pteridinas/síntese química , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proliferação de Células/efeitos dos fármacos , Relação Estrutura-Atividade , Caseína Quinase Idelta/antagonistas & inibidores , Caseína Quinase Idelta/metabolismo , Caseína Quinase 1 épsilon/antagonistas & inibidores , Caseína Quinase 1 épsilon/metabolismo , Linhagem Celular TumoralRESUMO
The human genome encodes two active Vaccinia-related protein kinases (VRK), VRK1 and VRK2. These proteins have been implicated in a number of cellular processes and linked to a variety of tumors. However, understanding the cellular role of VRKs and establishing their potential use as targets for therapeutic intervention has been limited by the lack of tool compounds that can specifically modulate the activity of these kinases in cells. Here we identified BI-D1870, a dihydropteridine inhibitor of RSK kinases, as a promising starting point for the development of chemical probes targeting the active VRKs. We solved co-crystal structures of both VRK1 and VRK2 bound to BI-D1870 and of VRK1 bound to two broad-spectrum inhibitors. These structures revealed that both VRKs can adopt a P-loop folded conformation, which is stabilized by different mechanisms on each protein. Based on these structures, we suggest modifications to the dihydropteridine scaffold that can be explored to produce potent and specific inhibitors towards VRK1 and VRK2.
Assuntos
Antineoplásicos/química , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pteridinas/química , Sequência de Aminoácidos , Antineoplásicos/farmacologia , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Genoma Humano , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Cinética , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Pteridinas/farmacologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Vaccinia virus/genética , Vaccinia virus/metabolismoRESUMO
Steady-state and time-resolved studies on quenching of excited states of pterin (Ptr) and lumazine (Lum) in the presence of iodide in aqueous solution have been performed. In contrast to the typical iodide enhancement in the triplet state population, iodide promotes a fast non-radiative T1â S0 transition for both Ptr and Lum. In this work, we present evidence for the effective iodide-induced deactivation of singlet and triplet excited states, with rate constants close to the diffusion-controlled limit (between 3 × 10(9) M(-1) s(-1) and 1 × 10(10) M(-1) s(-1)). The longer lifetimes of the triplet excited states over the singlet excited states increase the probability of deactivation (k(T)(q)τ(0)(T)â«k(S)(q)τ(0)(S)). Therefore, at micromolar concentrations of iodide, where the deactivation of the singlet excited state is negligible, an efficient deactivation of the triplet excited states is observed. This selective deactivation of the excited triplet state is an analytical tool for the study of photosensitized reactions where pteridines are involved.
Assuntos
Pteridinas/química , Iodetos/química , Lasers , Fotólise , Teoria Quântica , Espectrometria de FluorescênciaRESUMO
UV-A (320-400 nm) and UV-B (280-320 nm) radiation causes damage to DNA and other biomolecules through reactions induced by different endogenous or exogenous photosensitizers. Lumazines are heterocyclic compounds present in biological systems as biosynthetic precursors and/or products of metabolic degradation. The parent and unsubstituted compound called lumazine (pteridine-2,4(1,3H)-dione; Lum) is able to act as photosensitizer through electron transfer-initiated oxidations. To get further insight into the mechanisms involved, we have studied in detail the oxidation of 2'-deoxyadenosine 5'-monophosphate (dAMP) photosensitized by Lum in aqueous solution. After UV-A or UV-B excitation of Lum and formation of its triplet excited state ((3)Lum*), three reaction pathways compete for the deactivation of the latter: intersystem crossing to singlet ground state, energy transfer to O(2), and electron transfer between dAMP and (3)Lum* yielding the corresponding pair of radical ions (LumË(-) and dAMPË(+)). In the following step, the electron transfer from LumË(-) to O(2) regenerates Lum and forms the superoxide anion (O(2)Ë(-)), which undergoes disproportionation into H(2)O(2) and O(2). Finally dAMPË(+) participates in subsequent reactions to yield products.
Assuntos
Processos Fotoquímicos , Fármacos Fotossensibilizantes/química , Pteridinas/química , Nucleotídeos de Desoxiadenina/química , Transporte de Elétrons , Concentração de Íons de Hidrogênio , Oxirredução , Solubilidade , Superóxidos/químicaRESUMO
UV radiation induces damages to the DNA molecule and its components through photosensitized reactions. Among these processes, photosensitized oxidations may occur through electron transfer or hydrogen abstraction (type I mechanism) and/or the production of singlet molecular oxygen ((1)O(2)) (type II mechanism). Lumazines are an important family of heterocyclic compounds present in biological systems as biosynthetic precursors and/or products of metabolic degradation. To evaluate the capability of lumazines to act as photosensitizers through type I mechanism, we have investigated the oxidation of 2'-deoxyadenosine 5'-monophosphate (dAMP) photosensitized by the specific compound called lumazine (pteridine-2,4(1,3H)-dione; Lum) in aqueous solutions under UV irradiation. The photochemical reactions were followed by UV/vis spectrophotometry, HPLC, electrochemical measurement of dissolved O(2), and an enzymatic method for H(2)O(2) determination. The effect of pH was evaluated and the participation of oxygen was investigated. In aerated solutions, oxidation of dAMP photoinduced by the acid form of Lum (pH 5.5) takes place through a type I mechanism, in which the excitation of Lum is followed by an electron transfer from dAMP molecule to the Lum triplet excited state. During the process, O(2) is consumed and H(2)O(2) is generated, whereas the photosensitizer is not consumed. In contrast, no evidence of a photochemical reaction induced by the basic form of Lum (pH 10.5) was observed.
Assuntos
Nucleotídeos de Desoxiadenina/química , Oxidantes Fotoquímicos/química , Fármacos Fotossensibilizantes/química , Pteridinas/química , Cromatografia Líquida de Alta Pressão , Dano ao DNA/efeitos da radiação , Peróxido de Hidrogênio/análise , Concentração de Íons de Hidrogênio , Modelos Químicos , Oxigênio/análise , Processos Fotoquímicos , Fotoquímica , Oxigênio Singlete/química , Espectrofotometria Ultravioleta , Raios UltravioletaRESUMO
There are major differences between the structures of human dihydrofolate reductase (hDHFR) and Mycobacterium tuberculosis dihydrofolate reductase (mtDHFR). These differences may allow the design of more selective mtDHFR inhibitors. In this paper, we have used docking approaches to study the binding orientations and predict binding affinities of 2,4-diamino-5-methyl-5-deazapteridines derivatives in both hDHFR and mtDHFR. Our results of molecular docking combined with experimental data for inhibition of the human and mycobacterial dihydrofolate reductases suggest the presence of empty spaces around the 2,4-diaminodeazapteridine and N10-phenyl rings in the mtDHFR active site that are not found in the hDHFR-bound structures. Preparation of new analogs with substituents attached to C7 of the pteridine nucleus and positions 3 and 4 of the N10-phenyl group should increase the affinity and selectivity for mtDHFR.
Assuntos
Antagonistas do Ácido Fólico/metabolismo , Mycobacterium tuberculosis/enzimologia , Pteridinas/metabolismo , Tetra-Hidrofolato Desidrogenase/metabolismo , Antituberculosos/química , Antituberculosos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Simulação por Computador , Antagonistas do Ácido Fólico/química , Humanos , Modelos Moleculares , Ligação Proteica/fisiologia , Pteridinas/químicaRESUMO
Trinidad guppies (Poecilia reticulata) are distributed along an environmental gradient in carotenoid availability that limits the carotenoid content of the orange spots of males. The amount of synthetic red pteridines (drosopterins) in the orange spots covaries with the carotenoid content, such that the ratio of the two types of pigments is roughly conserved across streams. Carotenoids and drosopterins have different spectral properties and thus the ratio of the two types of pigments affects the shape of the orange spot reflectance spectrum. Geographic conservation of the carotenoid:drosopterin ratio suggests that males may be under selection to maintain a particular hue. We tested this hypothesis by comparing the pigmentation and coloration of guppies from six streams in the field to that of second-generation descendants of the same populations raised on three dietary carotenoid levels in the laboratory. The results show clearly that the geographic variation in drosopterin production is largely genetic and that the hue of the orange spots is conserved among populations in the field, relative to the laboratory diet groups. This is a countergradient pattern because genetic differences between populations in drosopterin production mask the effect of carotenoid availability on the hue of the orange spots. The potential for countergradient sexual selection to contribute to reproductive isolation between populations is discussed.
Assuntos
Carotenoides/metabolismo , Poecilia/fisiologia , Pteridinas/metabolismo , Pigmentação da Pele/genética , Animais , Carotenoides/administração & dosagem , Cor , Dieta , Feminino , Variação Genética , Masculino , Fenótipo , Poecilia/genética , Poecilia/metabolismo , Pteridinas/química , Rios , Seleção Genética , Pigmentação da Pele/efeitos dos fármacos , Análise Espectral , Trinidad e TobagoRESUMO
The liquid chromatographic retention factors extrapolated to pure water, k'(w), for several 6,7-diaryl-pteridine derivatives in both an octadecylsilane (ODS) and an immobilized artificial membrane column (IAM.PC.DD2), using acetonitrile-aqueous buffer pH = 7.45 as mobile phase, were obtained. The logarithms of the k'(w) values in the IAM.PC.DD2 column, log k'(w) (IAM), show good correlation with the calculated values of the octanol-water partition coefficients, log P(o/w), showing that the chromatographic parameter can be used as lipophilicity descriptor for the studied pteridines. However, interactions other than the lipophilic ones seem to be involved in the ODS column. Previous studies have shown that pteridines have antihelmintic properties. In spite of the complexity of the studied biological system as compared with the chromatographic one, good correlation between the descriptors obtained in the IAM column and biological activity (expressed as the log of the inhibitory concentration required to obtain up to 50% in the reduction of population growth of nematodes, log IC(50)) was observed.
Assuntos
Anti-Helmínticos/química , Anti-Helmínticos/farmacologia , Pteridinas/química , Relação Quantitativa Estrutura-Atividade , TemperaturaRESUMO
The structures of the P cluster and cofactor cluster of nitrogenase are well-defined crystallographically. They have been obtained only by biosynthesis; their chemical synthesis remains a challenge. Synthetic routes are sought to the P cluster in the P(N) state in which two cuboidal Fe(3)S(3) units are connected by a mu(6)-S atom and two Fe-(mu(2)-S(Cys))-Fe bridges. A reaction scheme affording a Mo(2)Fe(6)S(9) cluster in molecular form having the topology of the P(N) cluster has been devised. Reaction of the single cubane [(Tp)MoFe(3)S(4)Cl(3)](1)(-) with PEt(3) gives [(Tp)MoFe(3)S(4)(PEt(3))(3)](1+) (2), which upon reduction with BH(4)(-) affords the edge-bridged all-ferrous double cubane [(Tp)(2)Mo(2)Fe(6)S(8)(PEt(3))(4)] (4) (Tp = tris(pyrazolylhydroborate(1-)). Treatment of 4 with 3 equiv of HS(-) produces [(Tp)(2)Mo(2)Fe(6)S(9)(SH)(2)](3)(-) (7) as the Et(4)N(+) salt in 86% yield. The structure of 7 is built of two (Tp)MoFe(3)(mu(3)-S)(3) cuboidal fragments bridged by two mu(2)-S atoms and one mu(6)-S atom in an arrangement of idealized C(2) symmetry. The cluster undergoes three one-electron oxidation reactions and is oxidatively cleaved by p-tolylthiol to [(Tp)MoFe(3)S(4)(S-p-tol)(3)](2)(-) and by weak acids to [(Tp)MoFe(3)S(4)(SH)(3)](2-). The cluster core of 7 has the bridging pattern [Mo(2)Fe(6)(mu(2)-S)(2)(mu(3)-S)(6)(mu(6)-S)](1+) with the probable charge distribution [Mo(3+)(2)Fe(2+)(5)Fe(3+)S(9)](1+). Cluster 7 is a topological analogue of the P(N) cluster but differs in having two heteroatoms and two Fe-(mu(2)-S)-Fe instead of two Fe-(mu(2)-S(Cys))-Fe bridges. A best-fit superposition of the two cluster cores affords a weighted rms deviation in atom positions of 0.38 A. Cluster 7 is the first molecular topological analogue of the P(N) cluster. This structure had been prepared previously only as a fragment of complex high-nuclearity Mo-Fe-S clusters.
Assuntos
Materiais Biomiméticos/síntese química , Coenzimas , Ferro/química , Molibdênio/química , Nitrogenase/química , Enxofre/química , Materiais Biomiméticos/química , Cristalografia por Raios X , Espectroscopia de Ressonância Magnética , Metaloproteínas/química , Modelos Moleculares , Estrutura Molecular , Cofatores de Molibdênio , Compostos Organometálicos/síntese química , Compostos Organometálicos/química , Pteridinas/química , Compostos de Selênio/químicaRESUMO
Biopterin, isoxanthopterin and 6-pterincarboxylic acid were identified in the head of the malaria vector mosquito Anopheles albimanus Weidemann (Diptera: Culicidae) by HPLC. Total pteridine concentrations (TPC) were estimated in heads, body parts (BP: abdomen, legs and wings) and whole bodies of insectary-reared and field-collected females, by spectrofluorometry, to investigate whether they could be used for age determination. Pteridine concentrations diminished with age in both mosquito groups. TPC correlated with chronological age in insectary-reared sugar-fed females (heads: r2 = 0.35, BP: r2 = 0.34, P < 0.001), but lower correlation occurred in blood-fed females (heads: r2 = 0.22, BP: r2 = 0.27). TPC differed among females of the same age fed with blood at different times (P < 0.05), indicating that bloodmeals modify the diminution rate of pteridines with age. Nevertheless, a polynomial significant correlation was documented for TPC and the number of ovipositions (heads: r2 = 0.24, BP: r2 = 0.27, whole body: r2 = 0.52, P < 0.001) in insectary-reared mosquitoes. This correlation was lower in field-collected mosquitoes (heads: r2 = 0.14, BP: r2 = 0.10, P < 0.05), which showed a remarkable pteridine increase in one-parous females. The correlation of TPC in whole body with physiological age was much less (r2 = 0.03). These observations indicate that TPC determination by spectrofluorometry is not a reliable method to estimate the age of An. albimanus females from the feral population.