RESUMO
Hydroxycarbodenafil, an analogue of carbodenafil, was detected in a dietary supplement in China in 2020. However, previous reports have not identified some carbon signals from the piperazine ring in nuclear magnetic resonance (NMR) experiments. Because the compound contains an amide bond, the reaction was suggested to be characteristic of compounds with rotational isomers. Variable-temperature NMR is used to determine the rotational barrier between different conformations by changing the measurement temperature. Using this technique, we succeeded in obtaining the first distinct data, including the carbon signals of the piperazine ring in the NMR spectrum of hydroxycarbodenafil. We also confirmed that this technique could be applied to other carbodenafil analogues. Multi-stage mass spectrometry (MSn) measurements with a high-resolution mass spectrometer specific to the substructures were performed to develop a protocol for the structural determination of the carbodenafil analogues. In addition, hydroxycarbodenafil was analysed using X-ray crystallography, and its inhibitory activity against phosphodiesterase type 5 (PDE5) was measured. The IC50 value of the inhibitory activity of hydroxycarbodenafil for PDE5A1, a PDE5 isoform, of 2.9â¯nM was lower than the 4.5â¯nM for sildenafil, a positive control.
Assuntos
Espectroscopia de Ressonância Magnética , Inibidores da Fosfodiesterase 5 , Temperatura , Inibidores da Fosfodiesterase 5/química , Inibidores da Fosfodiesterase 5/análise , Inibidores da Fosfodiesterase 5/farmacologia , Espectroscopia de Ressonância Magnética/métodos , Cristalografia por Raios X/métodos , Espectrometria de Massas em Tandem/métodos , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/química , Piperazinas/química , Piperazinas/farmacologia , Piperazinas/análiseRESUMO
Phosphodiesterase-5 (PDE5) is responsible for regulating the concentration of the second messenger molecule cGMP by hydrolyzing it into 5'-GMP. PDE5 is implicated in erectile dysfunction and cardiovascular diseases. The substrate binding site in the catalytic domain of PDE5 is surrounded by several dynamic structural motifs (including the α14 helix, M-loop, and H-loop) that are known to switch between inactive and active conformational states via currently unresolved structural intermediates. We evaluated the conformational dynamics of these structural motifs in the apo state and upon binding of an allosteric inhibitor (evodiamine) or avanafil, a competitive inhibitor. We employed enhanced sampling-based replica exchange solute scaling (REST2) method, principal component analysis (PCA), time-lagged independent component analysis (tICA), molecular dynamics (MD) simulations, and well-tempered metadynamics simulations to probe the conformational changes in these structural motifs. Our results support a regulatory mechanism for PDE5, where the α14 helix alternates between an inward (lower activity) conformation and an outward (higher activity) conformation that is accompanied by the folding/unfolding of the α8' and α8â³ helices of the H-loop. When the allosteric inhibitor evodiamine is bound to PDE5, the inward (inactive) state of the α14 helix is preferred, thus preventing substrate access to the catalytic site. In contrast, competitive inhibitors of PDE5 block catalysis by occupying the active site accompanied by stabilization of the outward conformation of the α14 helix. Defining the conformational dynamics underlying regulation of PDE5 activation will be helpful in rational design of next-generation small molecules modulators of PDE5 activity.
Assuntos
GMP Cíclico , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/química , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/metabolismo , Sítios de Ligação , Domínio Catalítico , GMP Cíclico/químicaRESUMO
A ligand-based approach involving systematic modifications of a trisubstituted pyrazoline scaffold derived from the COX2 inhibitor, celecoxib, was used to develop novel PDE5 inhibitors. Novel pyrazolines were identified with potent PDE5 inhibitory activity lacking COX2 inhibitory activity. Compound d12 was the most potent with an IC50 of 1 nM, which was three times more potent than sildenafil and more selective with a selectivity index of >10,000-fold against all other PDE isozymes. Sildenafil inhibited the full-length and catalytic fragment of PDE5, while compound d12 only inhibited the full-length enzyme, suggesting a mechanism of enzyme inhibition distinct from sildenafil. The PDE5 inhibitory activity of compound d12 was confirmed in cells using a cGMP biosensor assay. Oral administration of compound d12 achieved plasma levels >1000-fold higher than IC50 values and showed no discernable toxicity after repeated dosing. These results reveal a novel strategy to inhibit PDE5 with unprecedented potency and isozyme selectivity.
Assuntos
Celecoxib/química , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/química , Inibidores da Fosfodiesterase 5/química , Pirazóis/química , Animais , Proteínas Sanguíneas/química , Proteínas Sanguíneas/metabolismo , Celecoxib/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/metabolismo , Desenho de Fármacos , Feminino , Meia-Vida , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microssomos Hepáticos/metabolismo , Inibidores da Fosfodiesterase 5/metabolismo , Ligação Proteica , Pirazóis/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Unapproved ingredients included in herbal medicines and dietary supplements have been detected as adulterated synthetic drugs used for erectile dysfunction. Extraction from a dietary supplement was performed to isolate the compounds by HPLC analysis. The structural characterization was confirmed using mass spectrometry (ESI-TOF/MS and LC-MS/MS), 1H NMR, and 13C NMR spectroscopy techniques. Results identified the thus-obtained compound to be sulfoaildenafil, a thioketone analogue of sildenafil. The biological activities of this active compound have been focused for the first time by the experimental point of view performance in vitro. The results revealed that sulfoaildenafil can affect the therapeutic level of nitric oxide through the upregulation of nitric oxide synthase and phosphodiesterase type 5 (PDE5) gene expressions. This bulk material, which displays structural similarity to sildenafil, was analyzed for the presence of a PDE5 inhibitor using a theoretical calculation. These unique features of the potential activity of PDE5 protein and its inhibitors, sildenafil and sulfoaildenafil, may play a key consideration for understanding the mode of actions and predicting the biological activities of PDE5 inhibitors.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/genética , Suplementos Nutricionais , Disfunção Erétil/tratamento farmacológico , Inibidores da Fosfodiesterase 5/química , Cromatografia Líquida de Alta Pressão , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/química , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/efeitos dos fármacos , Disfunção Erétil/patologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Medicina Herbária , Humanos , Espectroscopia de Ressonância Magnética , Masculino , Espectrometria de Massas , Modelos Moleculares , Estrutura Molecular , Inibidores da Fosfodiesterase 5/uso terapêutico , Piperazinas/química , Piperazinas/uso terapêutico , Citrato de Sildenafila/química , Citrato de Sildenafila/uso terapêutico , Sulfonas/química , Sulfonas/uso terapêuticoRESUMO
The elaboration of new small molecules that target phosphodiesterase enzymes (PDEs), especially those of type 5 (PDE5), is an interesting and emerging topic nowadays. A new series of heterocycle-based aminothiazoles were designed and synthesized from the key intermediate, 3-oxo-N-(thiazol-2-yl)butanamide (a PDE5 inhibitor that retains its amidic function), as an essential pharmacophoric moiety. The PDE5 inhibitors prevent the degradation of cyclic guanosine monophosphate, thereby causing severe hypotension as a marked side effect. Hence, an in vivo testing of the target compounds was conducted to verify its relation with arterial blood pressure. Utilizing sildenafil as the reference drug, Compounds 5, 10a, and 11b achieved 100% inhibitions of PDE5 without significantly lowering the mean arterial blood pressures (115.95 ± 2.91, 110.3 ± 2.84, and 78.3 ± 2.57, respectively). The molecular docking study revealed that the tested compounds exhibited docking poses that were similar to that of sildenafil (exploiting the amide functionality that interacted with GLN:817:A). The molecular shape and electrostatic similarity revealed a comparable physically achievable electrostatic potential with the reference drug, sildenafil. Therefore, these concomitant results revealed that the tested compounds exerted sildenafil-like inhibitory effects (although without its known drawbacks) on blood circulation, thus suggesting that the tested compounds might represent a cornerstone of beneficial drug candidates for the safe treatment for erectile dysfunction.
Assuntos
GMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/química , Desenho de Fármacos , Inibidores da Fosfodiesterase 5/química , Inibidores da Fosfodiesterase 5/farmacologia , Piridinas/química , Tiazóis/química , Humanos , Relação Estrutura-AtividadeRESUMO
Phosphodiesterase 5A enzyme has been the upcoming and promising target in hypertension management. In this research, reported 270 bioactive natural products having antihypertensive potential were selected and docked against PDE5A using vLife MDS 4.6 software. Based on docking score, π-stacking, H-bond and ionic interactions with PDE5A, 82 tricyclic compounds were selected for further study. Protein residue Gln817A was associated in H-boding, Leu804A in ionic interaction whereas Val782A and Phe820A were associated in π-stacking interaction with ligand. In silico docking studies resulted in discovery of oxygen containing naphthofuran and nitrogen and oxygen containing pyrano quinolizine tricyclic lead scaffolds as novel PDE5A inhibitors. Additionally, developed pharmacophore model suggested that one centre of hydrogen bond acceptor, one aromatic centre and two aliphatic centres are minimum pharmacophoric features required in the molecule so as to show sildenafil like activity. The identified lead scaffolds would provide novel platform for drug discovery of bioactive natural products.
Assuntos
Produtos Biológicos/química , Produtos Biológicos/farmacologia , Inibidores da Fosfodiesterase 5/química , Inibidores da Fosfodiesterase 5/farmacologia , Simulação por Computador , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/química , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/metabolismo , Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Ligação de Hidrogênio , Ligantes , Simulação de Acoplamento Molecular , Nitrogênio/química , Oxigênio/química , Quinolizinas/química , Citrato de Sildenafila/química , SoftwareRESUMO
In 1998, sildenafil was marketed as the first FDA-approved oral drug for the treatment of erectile dysfunction (ED). During the last two decades, the commercialization of other synthetic phosphodiesterase 5 (PDE5) inhibitors has been paralleled by the rise of remedies based on natural molecules from different chemical classes (flavonoids, polyphenols and alkaloids in general). In this work, a set of in silico tools were applied to study a panel of 30 natural compounds claimed to be effective against ED in the scientific literature or in folk medicine. First, pharmacokinetic properties were analysed to exclude the compounds lacking in specific drug-like features. Estimated binding energy for PDE5 and selectivity towards other PDE isoforms were then considered to highlight some promising molecules. Finally, a detailed structural investigation of the interaction pattern with PDE in comparison with sildenafil was conducted for the best performing compound of the set.
Assuntos
Inibidores da Fosfodiesterase 5/química , Inibidores da Fosfodiesterase 5/farmacologia , Sítios de Ligação , Produtos Biológicos/farmacologia , Produtos Biológicos/uso terapêutico , Simulação por Computador , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/química , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Disfunção Erétil/tratamento farmacológico , Humanos , Masculino , Medicina Tradicional , Inibidores da Fosfodiesterase 5/farmacocinética , Inibidores da Fosfodiesterase 5/uso terapêutico , Citrato de Sildenafila/farmacologiaRESUMO
Phosphodiesterase 5 (PDE5) is one of the most well-studied phosphodiesterases (PDEs) that specifically targets cGMP typically generated by nitric oxide (NO)-mediated activation of the soluble guanylyl cyclase. Given the crucial role of cGMP generated through the activation of this cellular signaling pathway in a variety of physiologically processes, pharmacological inhibition of PDE5 has been demonstrated to have several therapeutic applications including erectile dysfunction and pulmonary arterial hypertension. While they are designed to inhibit PDE5, the inhibitors show different affinities and specificities against all PDE subtypes. Additionally, they have been shown to induce allosteric structural changes in the protein. These are mostly attributed to their chemical structure and, therefore, binding interactions with PDE catalytic domains. Therefore, understanding how these inhibitors interact with PDE5 and the structural basis of their selectivity is critically important for the design of novel, highly selective PDE5 inhibitors. Here, we review the structure of PDE5, how its function is regulated, and discuss the clinically available inhibitors that target phosphodiesterase 5, aiming to better understand the structural bases of their affinity and specificity. We also discuss the therapeutic indications of these inhibitors and the potential of repurposing for a wider range of clinical applications.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/metabolismo , Inibidores da Fosfodiesterase 5/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Animais , Domínio Catalítico , GMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/química , Humanos , Isoenzimas , Inibidores da Fosfodiesterase 5/química , Fosforilação , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
Akt3 regulates mitochondrial content in endothelial cells through the inhibition of PGC-1α nuclear localization and is also required for angiogenesis. However, whether there is a direct link between mitochondrial function and angiogenesis is unknown. Here we show that Akt3 depletion in primary endothelial cells results in decreased uncoupled oxygen consumption, increased fission, decreased membrane potential, and increased expression of the mitochondria-specific protein chaperones, HSP60 and HSP10, suggesting that Akt3 is required for mitochondrial homeostasis. Direct inhibition of mitochondrial homeostasis by the model oxidant paraquat results in decreased angiogenesis, showing a direct link between angiogenesis and mitochondrial function. Next, in exploring functional links to PGC-1α, the master regulator of mitochondrial biogenesis, we searched for compounds that induce this process. We found that, sildenafil, a phosphodiesterase 5 inhibitor, induced mitochondrial biogenesis as measured by increased uncoupled oxygen consumption, mitochondrial DNA content, and voltage-dependent anion channel protein expression. Sildenafil rescued the effects on mitochondria by Akt3 depletion or pharmacological inhibition and promoted angiogenesis, further supporting that mitochondrial homeostasis is required for angiogenesis. Sildenafil also induces the expression of PGC-1 family member PRC and can compensate for PGC-1α activity during mitochondrial stress by an Akt3-independent mechanism. The induction of PRC by sildenafil depends upon cAMP and the transcription factor CREB. Thus, PRC can functionally substitute during Akt3 depletion for absent PGC-1α activity to restore mitochondrial homeostasis and promote angiogenesis. These findings show that mitochondrial homeostasis as controlled by the PGC family of transcriptional activators is required for angiogenic responses.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/química , Endotélio Vascular/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Inibidores da Fosfodiesterase 5/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Núcleo Celular/efeitos dos fármacos , Regulação da Expressão Gênica , Humanos , Mitocôndrias/patologia , Biogênese de Organelas , Consumo de Oxigênio , Fatores de Transcrição/genéticaRESUMO
Molecular recognition binding sites that specifically identify a target molecule are essential for life science research, clinical diagnoses, and therapeutic development. Corona phase molecular recognition is a technique introduced to generate synthetic recognition at the surface of a nanoparticle corona, but it remains an important question whether such entities can achieve the specificity of natural enzymes and receptors. In this work, we generate and screen a library of 24 amphiphilic polymers, preselected for molecular recognition and based on functional monomers including methacrylic acid, acrylic acid, and styrene, iterating upon a poly(methacrylic acid-co-styrene) motif. When complexed to a single-walled carbon nanotube, some of the resulting corona phases demonstrate binding specificity remarkably similar to that of phosphodiesterase type 5 (PDE5), an enzyme that catalyzes the hydrolysis of secondary messenger. The corona phase binds selectively to a PDE5 inhibitor, Vardenafil, as well as its molecular variant, but not to other potential off-target inhibitors. Our work herein examines the specificity and sensitivity of polymer "mutations" to the corona phase, as well as direct competitions with the native binding PDE5. Using structure perturbation, corona surface characterization, and molecular dynamics simulations, we show that the molecular recognition is associated with the unique three-dimensional configuration of the corona phase formed at the nanotube surface. This work conclusively shows that corona phase molecular recognition can mimic key aspects of biological recognition sites and drug targets, opening up possibilities for pharmaceutical and biological applications.
Assuntos
Materiais Biomiméticos , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5 , Nanotubos de Carbono/química , Sítios de Ligação , Materiais Biomiméticos/química , Materiais Biomiméticos/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/química , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/metabolismo , Simulação de Dinâmica Molecular , Dicloridrato de Vardenafila/química , Dicloridrato de Vardenafila/metabolismoRESUMO
Diabetic nephropathy (DN) is a leading cause of end-stage renal disease (ESRD). Hypertension increases kidney stress, which deteriorates function, and leads to peripheral renal vascular resistance. Long-term hypoperfusion promotes interstitial fibrosis and glomerular sclerosis, resulting in nephrosclerosis. Although hypertension and DN are frequent ESRD complications, relevant animal models remain unavailable. We generated a deoxycorticosterone acetate (DOCA)-salt hypertensive uni-nephrectomized (UNx) KKAy mouse model demonstrating hypertension, hyperglycemia, cardiac hypertrophy, kidney failure, increased urinary albumin creatinine ratio (UACR), and increased renal PDE4D and cardiac PDE5A mRNA levels. We hypothesized that the novel PDE4 selective inhibitor, compound A, and PDE5 inhibitor, sildenafil, exhibit nephroprotective, and cardioprotective effects in this new model. Compound A, sildenafil, and the angiotensin II receptor blocker, irbesartan, significantly reduced ventricular hypertrophy and pleural effusion volume. Meanwhile, compound A and sildenafil significantly suppressed the UACR, urinary kidney injury molecule-1, and monocyte chemoattractant protein-1 levels, as well as that of renal pro-fibrotic marker mRNAs, including collagen 1A1, fibronectin, and transforming growth factor-beta (TGF-ß). Moreover, compound A significantly suppressed TGF-ß-induced pro-fibrotic mRNA expression in vitro in all major kidney lesions, including within the glomerular mesangial region, podocytes, and epithelial region. Hence, PDE4 and PDE5 inhibitors may be promising treatments, in combination with irbesartan, for DN with hypertension as they demonstrate complementary mechanisms.
Assuntos
Cardiomegalia/tratamento farmacológico , Desoxicorticosterona/toxicidade , Hiperglicemia/tratamento farmacológico , Hipertensão/tratamento farmacológico , Inibidores da Fosfodiesterase 5/farmacologia , Insuficiência Renal/tratamento farmacológico , Citrato de Sildenafila/farmacologia , Acetatos/farmacologia , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Cardiomegalia/induzido quimicamente , Cardiomegalia/enzimologia , Cardiomegalia/patologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/química , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/química , Feminino , Hiperglicemia/induzido quimicamente , Hiperglicemia/enzimologia , Hiperglicemia/patologia , Hipertensão/induzido quimicamente , Hipertensão/enzimologia , Hipertensão/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mineralocorticoides/toxicidade , Insuficiência Renal/induzido quimicamente , Insuficiência Renal/enzimologia , Insuficiência Renal/patologia , Cloreto de Sódio/toxicidade , Tiramina/análogos & derivados , Tiramina/farmacologiaRESUMO
Alzheimer's disease (AD) is characterized by the progressive disturbance in cognition and affects approximately 36 million people, worldwide. However, the drugs used to treat this disease are only moderately effective and do not alter the course of the neurodegenerative process. This is because the pathogenesis of AD is mainly associated with oxidative stress, and current drugs only target two enzymes involved in neurotransmission. Therefore, the present study sought to identify potential multitarget compounds for enzymes that are directly or indirectly involved in the oxidative pathway, with minimal side effects, for AD treatment. A set of 159 lignans were submitted to studies of QSAR and molecular docking. A combined analysis was performed, based on ligand and structure, followed by the prediction of absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties. The results showed that the combined analysis was able to select 139 potentially active and multitarget lignans targeting two or more enzymes, among them are c-Jun N-terminal kinase 3 (JNK-3), protein tyrosine phosphatase 1B (PTP1B), nicotinamide adenine dinucleotide phosphate oxidase 1 (NOX1), NADPH quinone oxidoreductase 1 (NQO1), phosphodiesterase 5 (PDE5), nuclear factor erythroid 2-related factor 2 (Nrf2), cycloxygenase 2 (COX-2), and inducible nitric oxide synthase (iNOS). The authors conclude that compounds (06) austrobailignan 6, (11) anolignan c, (19) 7-epi-virolin, (64) 6-[(2R,3R,4R,5R)-3,4-dimethyl-5-(3,4,5-trimethoxyphenyl)oxolan-2-yl]-4-methoxy-1,3-benzodioxole, (116) ococymosin, and (135) mappiodoinin b have probabilities that confer neuroprotection and antioxidant activity and represent potential alternative AD treatment drugs or prototypes for the development of new drugs with anti-AD properties.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Avaliação Pré-Clínica de Medicamentos , Lignanas/análise , Lignanas/uso terapêutico , Interface Usuário-Computador , Algoritmos , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/química , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/metabolismo , Bases de Dados de Compostos Químicos , Humanos , Ligação de Hidrogênio , Lignanas/química , Simulação de Acoplamento Molecular , Proteína Tirosina Fosfatase não Receptora Tipo 1/química , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Relação Quantitativa Estrutura-Atividade , Curva ROC , TermodinâmicaRESUMO
Clinical use of phosphodiesterase-5 (PDE5) inhibitors is limited by several side effects due to weak isoform selectivity. Herein, a unique allosteric pocket of PDE5 is identified by molecular modeling and structural biology, which enables the discovery of highly selective PDE5 inhibitors from natural product evodiamine (EVO). The crystal structure of PDE5 with bound EVO derivative (S)-7e revealed that binding of (S)-7e to the novel allosteric pocket induced dramatic conformation changes in the H-loop with a maximum 24 Å movement of their Cα atoms. This movement directly blocks the binding of substrate/inhibitors to the PDE5 active site, which is different from all traditional PDE5 inhibitors such as sildenafil, tadalafil, and vardenafil. These derivatives showed >570-fold selectivity over PDE6C and PDE11A and achieved potent efficacy for the effective treatment of pulmonary hypertension in vivo.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/metabolismo , Inibidores da Fosfodiesterase 5/metabolismo , Quinazolinas/metabolismo , Sítio Alostérico , Sequência de Aminoácidos , Animais , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/química , Descoberta de Drogas , Masculino , Camundongos , Simulação de Acoplamento Molecular , Inibidores da Fosfodiesterase 5/química , Inibidores da Fosfodiesterase 5/farmacocinética , Ligação Proteica , Quinazolinas/química , Quinazolinas/farmacocinética , Ratos Sprague-Dawley , Alinhamento de Sequência , Relação Estrutura-AtividadeRESUMO
Phosphodiesterase 5A1 (PDE5) is a key target for treating cardiovascular diseases and erectile dysfunction. Here, we report the crystal structure of PDE5 complexed with the sole second generation drug avanafil. Analysis of protein-drug interactions revealed the structural basis of avanafil's superior isoform selectivity. Moreover, a halogen bonding was observed between avanafil and a backbone carbonyl oxygen of an adjacent α-helix, whose contribution to inhibitory potency illustrates the feasibility of exploiting α-helix backbone in structure-based drug design.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/química , Inibidores da Fosfodiesterase 5/farmacologia , Pirimidinas/farmacologia , Cristalografia por Raios X , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/metabolismo , Desenho de Fármacos , Humanos , Simulação de Acoplamento Molecular , Conformação Proteica/efeitos dos fármacos , Conformação Proteica em alfa-Hélice/efeitos dos fármacos , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismoRESUMO
We report that two widely-used drugs for erectile dysfunction, tadalafil and vardenafil, trigger bone gain in mice through a combination of anabolic and antiresorptive actions on the skeleton. Both drugs were found to enhance osteoblastic bone formation in vivo using a unique gene footprint and to inhibit osteoclast formation. The target enzyme, phosphodiesterase 5A (PDE5A), was found to be expressed in mouse and human bone as well as in specific brain regions, namely the locus coeruleus, raphe pallidus, and paraventricular nucleus of the hypothalamus. Localization of PDE5A in sympathetic neurons was confirmed by coimmunolabeling with dopamine ß-hydroxylase, as well as by retrograde bone-brain tracing using a sympathetic nerve-specific pseudorabies virus, PRV152. Both drugs elicited an antianabolic sympathetic imprint in osteoblasts, but with net bone gain. Unlike in humans, in whom vardenafil is more potent than tadalafil, the relative potencies were reversed with respect to their osteoprotective actions in mice. Structural modeling revealed a higher binding energy of tadalafil to mouse PDE5A compared with vardenafil, due to steric clashes of vardenafil with a single methionine residue at position 806 in mouse PDE5A. Collectively, our findings suggest that a balance between peripheral and central actions of PDE5A inhibitors on bone formation together with their antiresorptive actions specify the osteoprotective action of PDE5A blockade.
Assuntos
Disfunção Erétil/tratamento farmacológico , Osteogênese/efeitos dos fármacos , Osteoporose/tratamento farmacológico , Inibidores da Fosfodiesterase 5/farmacologia , Envelhecimento/fisiologia , Animais , Densidade Óssea/efeitos dos fármacos , Densidade Óssea/fisiologia , Osso e Ossos/citologia , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/metabolismo , Encéfalo/citologia , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Diferenciação Celular/efeitos dos fármacos , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/química , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/metabolismo , Reposicionamento de Medicamentos , Disfunção Erétil/complicações , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Modelos Animais , Modelos Moleculares , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Osteoblastos/efeitos dos fármacos , Osteoblastos/fisiologia , Osteoclastos/efeitos dos fármacos , Osteoclastos/fisiologia , Osteoporose/complicações , Fraturas por Osteoporose/etiologia , Fraturas por Osteoporose/prevenção & controle , Inibidores da Fosfodiesterase 5/química , Inibidores da Fosfodiesterase 5/uso terapêutico , Cultura Primária de Células , Tadalafila/química , Tadalafila/farmacologia , Tadalafila/uso terapêutico , Dicloridrato de Vardenafila/química , Dicloridrato de Vardenafila/farmacologia , Dicloridrato de Vardenafila/uso terapêuticoRESUMO
A rapid, smart and sensitive first derivative spectrofluorimetric method has been carried out for the simultaneous estimation of avanafil and tadalafil either in their pure form, tablet dosage form or spiked human plasma. The measurements of normal emission spectra or synchronous fluorescence intensity of both drugs show severe overlap which hindered their determination using normal fluorescence or synchronous intensity. Therefore, a highly sensitive first derivative synchronous fluorescence procedure was used to resolve this overlap. The method is based upon measurement of the amplitude of the first derivative of synchronous fluorescence intensity of both drugs at Δλ = 70 nm and at suitable wavelength of 396 nm and 364 nm for avanafil and tadalafil, respectively. Under the optimum conditions, the linear determination ranges are 50-1800 and 5-400 ng mL-1 with a detection limit of 12.93 and 1.46 ng mL-1 for avanafil and tadalafil, respectively. A response surface methodology was used for optimization using D-optimal design which can be used for determination of the exact optimum parameters specifically designed for this method. In addition; it is a good way to graphically clarify the relationship between various experimental variables and the synchronous fluorescence intensity.
Assuntos
Química Farmacêutica/métodos , Pirimidinas/análise , Espectrometria de Fluorescência , Tadalafila/análise , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/química , Fluorescência , Humanos , Modelos Lineares , Plasma/química , Reprodutibilidade dos Testes , ComprimidosRESUMO
The natural product icariin inhibits human phosphodiesterase-5 (PDE5) and represents a unique pharmacophore for treating erectile dysfunction, pulmonary hypertension, and other diseases. In this study, we explore the available icariin-derived chemical scaffolds through medicinal chemistry to develop novel icariin PDE5 inhibitors with improved potency and specificity. We synthesized six novel semi-synthetic icariin analogs as well as three naturally occurring icariin analogs, and characterized the structure-activity relationship in the context of human PDE5 inhibition using in vitro enzyme inhibition and kinetics assays and molecular modeling. Mammalian-cell-based assays and in vitro enzyme inhibition assays against human PDE6C further helped to identify the most potent and selective icariin analogs. Our results reveal the synergistic contribution of functional groups at the C3 and C7 positions of the icariin backbone towards PDE5 inhibition. Whereas a hydrophobic and flexible alkanol group at the C7 position is sufficient to enhance icariin analog potency, combining this group with a hydrophilic sugar group at the C3 position leads to further enhancement of potency and promotes specificity towards PDE5 versus PDE6C. In particular, compounds 3 and 7 exhibit Ki values of 0.036 ± 0.005 µM and 0.036 ± 0.007 µM towards PDE5 respectively, which are approaching those of commercial PDE5 inhibitors, and can effectively reduce GMP levels in cultured human BJ-hTERT cells. This study identifies novel icariin analogs as potent and selective PDE5 inhibitors poised to become lead compounds for further pharmaceutical development.
Assuntos
GMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/metabolismo , Flavonoides/farmacologia , Inibidores da Fosfodiesterase 5/farmacologia , Animais , Biocatálise/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , GMP Cíclico/química , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/química , Disfunção Erétil/tratamento farmacológico , Disfunção Erétil/metabolismo , Disfunção Erétil/fisiopatologia , Flavonoides/síntese química , Flavonoides/química , Humanos , Hipertensão Pulmonar/tratamento farmacológico , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/fisiopatologia , Masculino , Modelos Químicos , Estrutura Molecular , Inibidores da Fosfodiesterase 5/síntese química , Inibidores da Fosfodiesterase 5/química , Relação Estrutura-AtividadeRESUMO
In the current study, pyroglutamic acid (pGlu), a natural amino acid derivative, has efficiently inhibited the catalytic activities of three important enzymes, namely: Human recombinant phosphodiesterase-5A1 (PDE5A1), human angiotensin-converting enzyme (ACE), and urease. These enzymes were reported to be associated with several important clinical conditions in humans. Radioactivity-based assay, spectrophotometric-based assay, and an Electrospray Ionization-Mass Spectrometry-based method were employed to ascertain the inhibitory actions of pGlu against PDE5A1, ACE, and urease, respectively. The results unveiled that pGlu potently suppressed the activity of PDE5A1 (half-maximal inhibitory concentration; IC50 = 5.23 µM) compared with that of standard drug sildenafil citrate (IC50 = 7.14 µM). Moreover, pGlu at a concentration of 20 µg/mL was found to efficiently inhibit human ACE with 98.2% inhibition compared with that of standard captopril (99.6%; 20 µg/mL). The urease-catalyzed reaction was also remarkably inactivated by pGlu and standard acetohydroxamic acid with IC50 values of 1.8 and 3.9 µM, respectively. Remarkably, the outcome of in vitro cytotoxicity assay did not reveal any significant cytotoxic properties of pGlu against human cervical carcinoma cells and normal human fetal lung fibroblast cells. In addition to in vitro assays, molecular docking analyses were performed to corroborate the outcomes of in vitro results with predicted structure-activity relationships. In conclusion, pGlu could be presented as a natural and multifunctional agent with promising applications in the treatment of some ailments connected with the above-mentioned anti-enzymatic properties.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/metabolismo , Peptidil Dipeptidase A/metabolismo , Ácido Pirrolidonocarboxílico/química , Urease/metabolismo , Sítios de Ligação , Captopril/química , Captopril/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/química , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/genética , Humanos , Ácidos Hidroxâmicos/antagonistas & inibidores , Ácidos Hidroxâmicos/metabolismo , Concentração Inibidora 50 , Simulação de Acoplamento Molecular , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/genética , Estrutura Terciária de Proteína , Ácido Pirrolidonocarboxílico/metabolismo , Ácido Pirrolidonocarboxílico/toxicidade , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Citrato de Sildenafila/química , Citrato de Sildenafila/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Espectrofotometria , Relação Estrutura-Atividade , Urease/antagonistas & inibidoresRESUMO
Due to the exceptional wide range in biochemical activities of natural plant products, Spondias mombim L. are attaining a new height because they present great prospects for drug advancement. This research was designed to analyze the pharmaceutical properties of S. mombim L. ethyl acetate fraction (SMEAF) on key enzymes relevant to erectile and cognitive dysfunction. SMEAF inhibitory activities of the specified enzymes were determined spectrophotometrically. Chemical profile of SMEAF were assessed by HPLC/MS analysis. Thereafter, molecular docking of the studied enzymes with chlorogenic acid, lutein, and zeaxanthin were carried out using PATCHDOCK. SMEAF had remarkable enzyme inhibitory effects against phosphodiesterase-5 (PDE-5), arginase, angiotensin I-converting enzyme (ACE), cholinesterase, monoamine oxidase A (MAO), ecto-5' nucleotidase (E-NTDase), tyrosinase, and stimulated sodium-potassium ATPase (Na+/K+-ATPase) activities. HPLC/MS analysis revealed that phenolics and carotenoids were major components in these fraction notably, chlorogenic acid, lutein, and zeaxanthin. Our results suggested that SMEAF could be explored as phytopharmaceuticals. PRACTICAL APPLICATIONS: Spondias mombim L. are cooked as green vegetable with enormous medicinal value probably due to its polyphenols with potent antioxidant activity. Furthermore, the leaves could also be useful for therapeutic purposes against erectile dysfunction and central nervous system disorders.
Assuntos
Doença de Alzheimer/enzimologia , Anacardiaceae/química , Disfunção Erétil/enzimologia , Extratos Vegetais/química , Inibidores da Enzima Conversora de Angiotensina/química , Animais , Arginase/antagonistas & inibidores , Arginase/química , Inibidores da Colinesterase/química , Colinesterases/química , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/química , Inibidores Enzimáticos/química , Humanos , Masculino , Simulação de Acoplamento Molecular , Monoaminoxidase/química , Inibidores da Monoaminoxidase/química , Monofenol Mono-Oxigenase/antagonistas & inibidores , Monofenol Mono-Oxigenase/química , Peptidil Dipeptidase A/química , Inibidores da Fosfodiesterase 5/química , Folhas de Planta/química , Ratos , Ratos WistarRESUMO
Targeting chokepoint enzymes in metabolic pathways has led to new drugs for cancers, autoimmune disorders and infectious diseases. This is also a cornerstone approach for discovery and development of anthelmintics against nematode and flatworm parasites. Here, we performed omics-driven knowledge-based identification of chokepoint enzymes as anthelmintic targets. We prioritized 10 of 186 phylogenetically conserved chokepoint enzymes and undertook a target class repurposing approach to test and identify new small molecules with broad spectrum anthelmintic activity. First, we identified and tested 94 commercially available compounds using an in vitro phenotypic assay, and discovered 11 hits that inhibited nematode motility. Based on these findings, we performed chemogenomic screening and tested 32 additional compounds, identifying 6 more active hits. Overall, 6 intestinal (single-species), 5 potential pan-intestinal (whipworm and hookworm) and 6 pan-Phylum Nematoda (intestinal and filarial species) small molecule inhibitors were identified, including multiple azoles, Tadalafil and Torin-1. The active hit compounds targeted three different target classes in humans, which are involved in various pathways, including carbohydrate, amino acid and nucleotide metabolism. Last, using representative inhibitors from each target class, we demonstrated in vivo efficacy characterized by negative effects on parasite fecundity in hamsters infected with hookworms.