RESUMO
Cytochrome P450 enzymes (CYPs) play a crucial role in the metabolism and synthesis of various compound classes. While drug-metabolizing CYP enzymes are frequently investigated as anti-targets, the inhibition of CYP enzymes involved in adrenal steroidogenesis is not well studied. The steroidogenic enzyme CYP17A1 is a dual-function enzyme catalyzing hydroxylase and lyase reactions relevant for the biosynthesis of adrenal glucocorticoids and androgens. Inhibition of CYP17A1-hydroxylase leads to pseudohyperaldosteronism with subsequent excessive mineralocorticoid receptor activation, hypertension and hypokalemia. In contrast, specific inhibition of the lyase function might be beneficial for the treatment of prostate cancer by decreasing adrenal androgen levels. This study combined in silico and in vitro methods to identify drugs inhibiting CYP17A1. The most potent CYP17A1 inhibitors identified are serdemetan, mocetinostat, nolatrexed, liarozole, and talarozole. While some of these drugs are currently under investigation for the treatment of various cancers, their potential for the treatment of prostate cancer is yet to be explored. The DrugBank database was screened for CYP17A1 inhibitors, to increase the awareness for the risk of drug-induced pseudohyperaldosteronism and to highlight drugs so far unknown for their potential to cause side effects resulting from CYP17A1 inhibition.
Assuntos
Simulação por Computador , Esteroide 17-alfa-Hidroxilase , Esteroide 17-alfa-Hidroxilase/antagonistas & inibidores , Esteroide 17-alfa-Hidroxilase/metabolismo , Humanos , Masculino , Simulação de Acoplamento MolecularRESUMO
In this research article, we report on the strengthening of a non-classical hydrogen bond (C-H···O) by introducing electron withdrawing groups at the carbon atom. The approach is demonstrated on the example of derivatives of the physiological E-selectin ligand sialyl Lewisx (1, sLex). Its affinity is mainly due to a beneficial entropy term, which is predominantly caused by the pre-organization of sLex in its binding conformation. We have shown, that among the elements responsible for the pre-organization, the stabilization by a non-classical hydrogen bond between the H-C5 of l-fucose and the ring oxygen O5 of the neighboring d-galactose moiety is essential and yields 7.4 kJ mol-1. This effect could be further strengthened by replacing l-fucose by 6,6,6-trifluoro-l-fucose leading to an improved non-classical H-bond of 14.9 kJ mol-1, i.e., an improved pre-organization in the bioactive conformation. For a series of glycomimetics of sLex (1), this outcome could be confirmed by high field NMR-shifts of the H-C5Fuc, by X-ray diffraction analysis of glycomimetics co-crystallized with E-selectin as well as by isothermal titration calorimetry. Furthermore, the electron-withdrawing character of the CF3-group beneficially influences the pharmacokinetic properties of sLex mimetics. Thus, acid-stability a prerequisite for gastrointestinal stability could be substantially improved.
RESUMO
The pregnane X receptor (PXR) is a ligand-activated regulator of cytochrome P450 (CYP)3A enzymes. Among the ligands of human PXR is hyperforin, a constituent of St John's wort (SJW) extracts and potent inducer of human CYP3A4. It was the aim of this study to compare the effect of hyperforin and SJW formulations controlled for its content on CYP3A23-3A1 in rats. Hyperiplant was used as it contains a high hyperforin content and Rebalance because it is controlled for a low hyperforin content. In silico analysis revealed a weak hyperforin-rPXR binding affinity, which was further supported in cell-based reporter gene assays showing no hyperforin-mediated reporter activation in presence of rPXR. However, cellular exposure to Hyperiplant and Rebalance transactivated the CYP3A reporter 3.8-fold and 2.8-fold, respectively, and they induced Cyp3a23-3a1 mRNA expression in rat hepatoma cells compared with control 48-fold and 18-fold, respectively. In Wistar rats treated for 10 days with 400 mg/kg of Hyperiplant, we observed 1.8 times the Cyp3a23-3a1 mRNA expression, a 2.6-fold higher CYP3A23-3A1 protein amount, and a 1.6-fold increase in activity compared with controls. For Rebalance we only observed a 1.8-fold hepatic increase of CYP3A23-3A1 protein compared with control animals. Even though there are differing effects on rCyp3a23-3a1/CYP3A23-3A1 in rat liver reflecting the hyperforin content of the SJW extracts, the modulation is most likely not linked to an interaction of hyperforin with rPXR. SIGNIFICANCE STATEMENT: Treatment with St John's wort (SJW) has been reported to affect CYP3A expression and activity in rats. Our comparative study further supports this finding but shows that the pregnane X receptor-ligand hyperforin is not the driving force for changes in rat CYP3A23-3A1 expression and function in vivo and in vitro. Importantly, CYP3A induction mimics findings in humans, but our results suggest that another so far unknown constituent of SJW is responsible for the expression- and function-modifying effects in rat liver.
Assuntos
Antineoplásicos , Hypericum , Ratos , Humanos , Animais , Citocromo P-450 CYP3A/metabolismo , Receptor de Pregnano X , Hypericum/metabolismo , Ligantes , Ratos Wistar , RNA Mensageiro , Extratos Vegetais/farmacologia , Extratos Vegetais/químicaRESUMO
Several drugs were found after their market approval to unexpectedly inhibit adrenal 11ß-hydroxylase (CYP11B1)-dependent cortisol synthesis. Known side-effects of CYP11B1 inhibition include hypertension and hypokalemia, due to a feedback activation of adrenal steroidogenesis, leading to supraphysiological concentrations of 11-deoxycortisol and 11-deoxycorticosterone that can activate the mineralocorticoid receptor. This results in potassium excretion and sodium and water retention, ultimately causing hypertension. With the risk known but usually not addressed in preclinical evaluation, this study aimed to identify drugs and drug candidates inhibiting CYP11B1. Two conceptually different virtual screening methods were combined, a pharmacophore based and an induced fit docking approach. Cell-free and cell-based CYP11B1 activity measurements revealed several inhibitors with IC50 values in the nanomolar range. Inhibitors include retinoic acid metabolism blocking agents (RAMBAs), azole antifungals, α2-adrenoceptor ligands, and a farnesyltransferase inhibitor. The active compounds share a nitrogen atom embedded in an aromatic ring system. Structure activity analysis identified the free electron pair of the nitrogen atom as a prerequisite for the drug-enzyme interaction, with its pKa value as an indicator of inhibitory potency. Another important parameter is drug lipophilicity, exemplified by etomidate. Changing its ethyl ester moiety to a more hydrophilic carboxylic acid group dramatically decreased the inhibitory potential, most likely due to less efficient cellular uptake. The presented work successfully combined different in silico and in vitro methods to identify several previously unknown CYP11B1 inhibitors. This workflow facilitates the identification of compounds that inhibit CYP11B1 and therefore pose a risk for inducing hypertension and hypokalemia.
Assuntos
Hipertensão , Hipopotassemia , Humanos , Hipertensão/induzido quimicamente , Hipertensão/tratamento farmacológico , Hipopotassemia/complicações , Esteroide 11-beta-Hidroxilase/metabolismo , EsteroidesRESUMO
The d-GlcNAc moiety in sialyl Lewisx (sLex, 1) acts predominantly as a linker to position the d-Gal and the l-Fuc moieties in the bioactive spatial orientation. The hypothesis has been made that the NHAc group of GlcNAc pushes the fucose underneath the galactose and, thus, contributes to the stabilization of the bioactive conformation of the core of sLex (1). To test this hypothesis, GlcNAc mimetics consisting of (R,R)-1,2-cyclohexanediols substituted with alkyl and aryl substituents adjacent to the linking position of the fucose moiety were synthesized. To explore a broad range of extended and spatially demanding R-groups, an enzymatic approach for the synthesis of 3-alkyl/aryl-1,2-cyclohexanediols (3b-n) was applied. These cyclohexanediol derivatives were incorporated into the sLex mimetics 2b-n. For analyzing the relationship of affinity and core conformation, a 1H NMR structural-reporter-group concept was applied. Thus, the chemical shift of H-C5Fuc proved to be a sensitive indicator for the degree of pre-organization of the core of this class of sLex mimetics and therefore could be used to quantify the contribution of the R-groups.
Assuntos
Fucose , Oligossacarídeos , Antígeno Sialil Lewis X , Oligossacarídeos/química , Fucose/química , Conformação Molecular , Espectroscopia de Ressonância MagnéticaRESUMO
A library of more than 2500 plant extracts was screened for activity on oncogenic signaling in melanoma cells. The ethyl acetate extract from the aerial parts of Artemisia argyi displayed pronounced inhibition of the PI3K/AKT pathway. Active compounds were tracked with the aid of HPLC-based activity profiling, and altogether 21 active compounds were isolated, including one novel dimerosequiterpenoid (1), one new disesquiterpenoid (2), three new guaianolides (3-5), 12 known sesquiterpenoids (6-17), and four known flavonoids (19-22). A new eudesmanolide derivative (13b) was isolated as an artifact formed by methanolysis. Compound 1 is the first adduct comprising a sesquiterpene lactone and a methyl jasmonate moiety. The absolute configurations of compounds 1 and 3-18 were established by comparison of their experimental and calculated ECD spectra. The absolute configuration for 2 was determined by X-ray diffraction analysis. Guaianolide 8 was the most potent sesquiterpene lactone, inhibiting the PI3K/AKT pathway with an IC50 value of 8.9 ± 0.9 µM.
Assuntos
Antineoplásicos , Artemisia , Lactonas , Melanoma , Fosfatidilinositol 3-Quinases , Compostos Fitoquímicos , Inibidores de Proteínas Quinases , Proteínas Proto-Oncogênicas c-akt , Sesquiterpenos , Artemisia/química , Lactonas/química , Lactonas/isolamento & purificação , Lactonas/farmacologia , Melanoma/enzimologia , Estrutura Molecular , Fosfatidilinositol 3-Quinases/metabolismo , Compostos Fitoquímicos/química , Compostos Fitoquímicos/isolamento & purificação , Compostos Fitoquímicos/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Sesquiterpenos/química , Sesquiterpenos/isolamento & purificação , Sesquiterpenos/farmacologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/isolamento & purificação , Inibidores de Proteínas Quinases/farmacologia , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacologiaRESUMO
11ß-hydroxysteroid dehydrogenase 2 (11ß-HSD2) converts active 11ß-hydroxyglucocorticoids to their inactive 11-keto forms, thereby preventing inappropriate mineralocorticoid receptor activation by glucocorticoids. Disruption of 11ß-HSD2 activity by genetic defects or inhibitors causes the syndrome of apparent mineralocorticoid excess (AME), characterized by hypokalemia, hypernatremia and hypertension. Recently, the azole antifungals itraconazole and posaconazole were identified to potently inhibit human 11ß-HSD2, and several case studies described patients with acquired AME. To begin to understand why this adverse drug effect was missed during preclinical investigations, the inhibitory potential of itraconazole, its main metabolite hydroxyitraconazole (OHI) and posaconazole against 11ß-HSD2 from human and three commonly used experimental animals was assessed. Whilst human 11ß-HSD2 was potently inhibited by all three compounds (IC50 values in the nanomolar range), the rat enzyme was moderately inhibited (1.5- to 6-fold higher IC50 values compared to human), and mouse and zebrafish 11ß-HSD2 were very weakly inhibited (IC50 values above 7 µM). Sequence alignment and application of newly generated homology models for human and mouse 11ß-HSD2 revealed significant differences in the C-terminal region and the substrate binding pocket. Exchange of the C-terminus and substitution of residues Leu170,Ile172 in mouse 11ß-HSD2 by the corresponding residues His170,Glu172 of the human enzyme resulted in a gain of sensitivity to itraconazole and posaconazole, resembling human 11ß-HSD2. The results provide an explanation for the observed species-specific 11ß-HSD2 inhibition by the studied azole antifungals. The obtained structure-activity relationship information should facilitate future assessments of 11ß-HSD2 inhibitors and aid choosing adequate animal models for efficacy and safety studies.
Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 2/antagonistas & inibidores , Antifúngicos/toxicidade , Inibidores Enzimáticos/toxicidade , Itraconazol/toxicidade , Triazóis/toxicidade , Proteínas de Peixe-Zebra/antagonistas & inibidores , 11-beta-Hidroxiesteroide Desidrogenase Tipo 2/química , 11-beta-Hidroxiesteroide Desidrogenase Tipo 2/genética , 11-beta-Hidroxiesteroide Desidrogenase Tipo 2/metabolismo , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Síndrome de Excesso Aparente de Minerolocorticoides/induzido quimicamente , Síndrome de Excesso Aparente de Minerolocorticoides/enzimologia , Conformação Proteica , Especificidade da Espécie , Relação Estrutura-Atividade , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/genéticaRESUMO
The ligand-binding domain of the androgen receptor (AR) is a target for drugs against prostate cancer and offers three distinct binding sites for small molecules. Drugs acting on the orthosteric hormone binding site suffer from resistance mechanisms that can, in the worst case, reverse their therapeutic effect. While many allosteric ligands targeting either the activation function-2 (AF-2) or the binding function-3 (BF-3) have been reported, their potential for simultaneous administration with currently prescribed antiandrogens was disregarded. Here, we report results of 60 µs molecular dynamics simulations to investigate combinations of orthosteric and allosteric AR antagonists. Our results suggest BF-3 inhibitors to be more suitable in combination with classical antiandrogens as opposed to AF-2 inhibitors based on binding free energies and binding modes. As a mechanistic explanation for these observations, we deduced a structural adaptation of helix-12 involved in the formation of the AF-2 site by classical AR antagonists. Additionally, the changes were accompanied by an expansion of the orthosteric binding site. Considering our predictions, the selective combination of AR-targeting compounds may improve the treatment of prostate cancer.
Assuntos
Antagonistas de Receptores de Andrógenos , Receptores Androgênicos , Antagonistas de Androgênios/farmacologia , Antagonistas de Receptores de Andrógenos/farmacologia , Sítios de Ligação , Humanos , Ligantes , Masculino , Simulação de Dinâmica MolecularRESUMO
Thyroid hormone receptors (TRs) play a critical role in human development, growth, and metabolism. Antagonists of TRs offer an attractive strategy to treat hyperthyroidism without the disadvantage of a delayed onset of drug action. While it is challenging to examine the atomistic behavior of TRs in a laboratory setting, computational methods such as molecular dynamics (MD) simulations have proven their value to elucidate ligand-induced conformational changes in nuclear receptors. Here, we performed MD simulations of TRα and TRß complexed to their native ligand triiodothyronine (T3) as well as several antagonists. Based on the examination of 27 µs MD trajectories, we showed how binding of these compounds influences various structural features of the receptors including the helicity of helices 3 and 10 as well as the location of helix-12. Helices 3 and 12 are known to mediate coactivator association required for downstream signaling, suggesting these changes to be the molecular basis for TR antagonism. A mechanistic analysis of the trajectories revealed an allosteric pathway between H3 and H12 to be responsible for the conformational adaptations. Even though a mechanistic understanding of conformational adaptations triggered by TR antagonists is important for the development of novel therapeutics, they have not been previously examined in detail as it was done here.
Assuntos
Receptores dos Hormônios Tireóideos , Glândula Tireoide , Humanos , Ligantes , Receptores beta dos Hormônios Tireóideos , Tri-IodotironinaRESUMO
Cytochrome P450 enzymes (CYPs) are the largest group of enzymes involved in human drug metabolism. Ligand tunnels connect their active site buried at the core of the membrane-anchored protein to the surrounding solvent environment. Recently, evidence of a superficial allosteric site, here denoted as hotspot 1 (H1), involved in the regulation of ligand access in a soluble prokaryotic CYP emerged. Here, we applied multi-scale computational modeling techniques to study the conservation and functionality of this allosteric site in the nine most relevant mammalian CYPs responsible for approximately 70% of drug metabolism. In total, we systematically analyzed over 44 µs of trajectories from conventional MD, cosolvent MD, and metadynamics simulations. Our bioinformatic analysis and simulations with organic probe molecules revealed the site to be well conserved in the CYP2 family with the exception of CYP2E1. In the presence of a ligand bound to the H1 site, we could observe an enlargement of a ligand tunnel in several members of the CYP2 family. Further, we could detect the facilitation of ligand translocation by H1 interactions with statistical significance in CYP2C8 and CYP2D6, even though all other enzymes except for CYP2C19, CYP2E1, and CYP3A4 presented a similar trend. As the detailed comprehension of ligand access and egress phenomena remains one of the most relevant challenges in the field, this work contributes to its elucidation and ultimately helps in estimating the selectivity of metabolic transformations using computational techniques.
Assuntos
Biologia Computacional/métodos , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/metabolismo , Mamíferos/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Sítio Alostérico , Animais , Cânfora 5-Mono-Oxigenase/química , Cânfora 5-Mono-Oxigenase/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Citocromo P-450 CYP2C8/química , Citocromo P-450 CYP2C8/metabolismo , Citocromo P-450 CYP2D6/química , Citocromo P-450 CYP2D6/metabolismo , Humanos , Modelos Moleculares , Simulação de Dinâmica Molecular , Conformação Proteica , Bibliotecas de Moléculas Pequenas/químicaRESUMO
Oxidative reactions catalyzed by Cytochrome P450 enzymes (CYPs), which constitute the most relevant group of drug-metabolizing enzymes, are enabled by their redox partner Cytochrome P450 reductase (CPR). Both proteins are anchored to the membrane of the endoplasmic reticulum and the CPR undergoes a conformational change in order to interact with the respective CYP and transfer electrons. Here, we conducted over 22 microseconds of molecular dynamics (MD) simulations in combination with protein-protein docking to investigate the conformational changes necessary for the formation of the CPR-CYP complex. While some structural features of the CPR and the CPR-CYP2D6 complex that we highlighted confirmed previous observations, our simulations revealed additional mechanisms for the conformational transition of the CPR. Unbiased simulations exposed a movement of the whole protein relative to the membrane, potentially to facilitate interactions with its diverse set of redox partners. Further, we present a structural mechanism for the susceptibility of the CPR to different redox states based on the flip of a glycine residue disrupting the local interaction network that maintains inter-domain proximity. Simulations of the CPR-CYP2D6 complex pointed toward an additional interaction surface of the FAD domain and the proximal side of CYP2D6. Altogether, this study provides novel structural insight into the mechanism of CPR-CYP interactions and underlying conformational changes, improving our understanding of this complex machinery Cytochrome P450 reductase; CPR; conformational; dynamicsrelevant for drug metabolism.
Assuntos
Simulação de Dinâmica Molecular , NADPH-Ferri-Hemoproteína Redutase/química , Sítios de Ligação , Citocromo P-450 CYP2D6/química , Citocromo P-450 CYP2D6/metabolismo , Flavina-Adenina Dinucleotídeo/química , Flavina-Adenina Dinucleotídeo/metabolismo , Humanos , Simulação de Acoplamento Molecular , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Ligação Proteica , Multimerização ProteicaRESUMO
The pandemic of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a serious global health threat. Since no specific therapeutics are available, researchers around the world screened compounds to inhibit various molecular targets of SARS-CoV-2 including its main protease (Mpro) essential for viral replication. Due to the high urgency of these discovery efforts, off-target binding, which is one of the major reasons for drug-induced toxicity and safety-related drug attrition, was neglected. Here, we used molecular docking, toxicity profiling, and multiple molecular dynamics (MD) protocols to assess the selectivity of 33 reported non-covalent inhibitors of SARS-CoV-2 Mpro against eight proteases and 16 anti-targets. The panel of proteases included SARS-CoV Mpro, cathepsin G, caspase-3, ubiquitin carboxy-terminal hydrolase L1 (UCHL1), thrombin, factor Xa, chymase, and prostasin. Several of the assessed compounds presented considerable off-target binding towards the panel of proteases, as well as the selected anti-targets. Our results further suggest a high risk of off-target binding to chymase and cathepsin G. Thus, in future discovery projects, experimental selectivity assessment should be directed toward these proteases. A systematic selectivity assessment of SARS-CoV-2 Mpro inhibitors, as we report it, was not previously conducted.
Assuntos
Antivirais/química , Antivirais/farmacologia , Tratamento Farmacológico da COVID-19 , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , SARS-CoV-2/efeitos dos fármacos , COVID-19/enzimologia , Proteases 3C de Coronavírus/antagonistas & inibidores , Proteases 3C de Coronavírus/química , Proteases 3C de Coronavírus/metabolismo , Descoberta de Drogas/métodos , Humanos , Simulação de Acoplamento Molecular/métodos , Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , SARS-CoV-2/enzimologiaRESUMO
The xenobiotic metabolizing enzyme CYP2D6 is the P450 cytochrome family member with the highest rate of polymorphism. This causes changes in the enzyme activity and specificity, which can ultimately lead to adverse reactions during drug treatment. To avoid or lower CYP-related toxicity risks, prediction of the most likely positions within a molecule where a metabolic reaction might occur is paramount. In order to obtain accurate predictions, it is crucial to understand all phenomena within the active site of the enzyme that contribute to an efficient substrate recognition and the subsequent catalytic reaction together with their relative weight within the overall thermodynamic context. This study aims to define the weight of the driving forces upon the C-H bond activation within CYP2D6 wild-type and a clinically relevant allelic variant with increased activity (CYP2D6*53) featuring two amino acid mutations in close vicinity of the heme. First, we investigated the steric and electrostatic complementarity of the substrate bufuralol using well-tempered metadynamics simulations with the aim to obtain the free energy profiles for each site of metabolism (SoM) within the different active sites. Second, the stereoelectronic complementarity was determined for each SoM within the two different active-site environments. Relying on the well-tempered metadynamics simulation energy profiles of each SoM, we identified the binding mode that was closest to the preferred transition-state geometry for efficient C-H bond activation. The binding modes were then used as starting structures for the quantum mechanics/molecular mechanics calculations performed to quantify the corresponding activation barriers. Our results show the relevance of the steric component in orienting the SoM in an energetically accessible position toward the heme. However, the corresponding intrinsic reactivity and electronic complementarity within the active site must be accurately evaluated in order to obtain a meaningful reaction prediction, from which the predominant SoM can be determined. The F120I mutation lowered the activation barrier for the major site and one of the minor SoMs. However, it had an impact neither on the CYP2D6 enantioselectivity preference of the oxidation reaction nor on the stereoselectivity from the substrate point of view.
Assuntos
Citocromo P-450 CYP2D6 , Simulação de Dinâmica Molecular , Domínio Catalítico , Citocromo P-450 CYP2D6/genética , Sistema Enzimático do Citocromo P-450 , Especificidade por SubstratoRESUMO
In a screening of an extract library from plants used in Traditional Chinese Medicine the MeOH extract of Toddalia asiatica inhibited proliferation of human primary T cells with an IC50 of 25.8 µg/mL. Activity in the extract was tracked by HPLC activity profiling, and a total of 15 compounds were characterized. Three compounds, toddalic acid (6) and both enantiomers (7a and 7b) of toddanolic acid (7), were new natural products, and two recently published compounds, (2'R)-toddalolactone 3'-O-ß-d-glucopyranoside (10) and (2'S)-toddalolactone 2'-O-ß-d-glucopyranoside (11), were described in detail for the first time. The absolute configurations of compounds 8, 9, 10, 12, 13, and 15 were determined by comparison of experimental and calculated ECD spectra. For glucosides 9 and 10, ECD data and chiral-phase HPLC of the aglycones after enzymatic hydrolysis confirmed the results. Nitidine chloride (4) inhibited proliferation of primary human T cells with an IC50 of 0.4 µM.
Assuntos
Imunossupressores/farmacologia , Extratos Vegetais/farmacologia , Rutaceae , Cumarínicos , Glucosídeos , Estrutura Molecular , Raízes de Plantas , EstereoisomerismoRESUMO
Nuclear receptors (NRs) are highly relevant drug targets in major indications such as oncologic, metabolic, reproductive, and immunologic diseases. However, currently, marketed drugs designed towards the orthosteric binding site of NRs often suffer from resistance mechanisms and poor selectivity. The identification of two superficial allosteric sites, activation function-2 (AF-2) and binding function-3 (BF-3), as novel drug targets sparked the development of inhibitors, while selectivity concerns due to a high conservation degree remained. To determine important pharmacophores and hydration sites among AF-2 and BF-3 of eight hormonal NRs, we systematically analyzed over 10 µ s of molecular dynamics simulations including simulations in explicit water and solvent mixtures. In addition, a library of over 300 allosteric inhibitors was evaluated by molecular docking. Based on our results, we suggest the BF-3 site to offer a higher potential for drug selectivity as opposed to the AF-2 site that is more conserved among the selected receptors. Detected similarities among the AF-2 sites of various NRs urge for a broader selectivity assessment in future studies. In combination with the Supplementary Material, this work provides a foundation to improve both selectivity and potency of allosteric inhibitors in a rational manner and increase the therapeutic applicability of this promising compound class.
Assuntos
Sítio Alostérico , Sítios de Ligação , Ligantes , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Receptores Citoplasmáticos e Nucleares/química , Regulação Alostérica/efeitos dos fármacos , Conformação Molecular , Ligação Proteica , Relação Quantitativa Estrutura-Atividade , Receptores Citoplasmáticos e Nucleares/metabolismoRESUMO
The rapid outbreak of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in China followed by its spread around the world poses a serious global concern for public health. To this date, no specific drugs or vaccines are available to treat SARS-CoV-2 despite its close relation to the SARS-CoV virus that caused a similar epidemic in 2003. Thus, there remains an urgent need for the identification and development of specific antiviral therapeutics against SARS-CoV-2. To conquer viral infections, the inhibition of proteases essential for proteolytic processing of viral polyproteins is a conventional therapeutic strategy. In order to find novel inhibitors, we computationally screened a compound library of over 606 million compounds for binding at the recently solved crystal structure of the main protease (Mpro) of SARS-CoV-2. A screening of such a vast chemical space for SARS-CoV-2 Mpro inhibitors has not been reported before. After shape screening, two docking protocols were applied followed by the determination of molecular descriptors relevant for pharmacokinetics to narrow down the number of initial hits. Next, molecular dynamics simulations were conducted to validate the stability of docked binding modes and comprehensively quantify ligand binding energies. After evaluation of potential off-target binding, we report a list of 12 purchasable compounds, with binding affinity to the target protease that is predicted to be more favorable than that of the cocrystallized peptidomimetic compound. In order to quickly advise ongoing therapeutic intervention for patients, we evaluated approved antiviral drugs and other protease inhibitors to provide a list of nine compounds for drug repurposing. Furthermore, we identified the natural compounds (-)-taxifolin and rhamnetin as potential inhibitors of Mpro. Rhamnetin is already commercially available in pharmacies.
Assuntos
Infecções por Coronavirus/enzimologia , Pneumonia Viral/enzimologia , Inibidores de Proteases/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Sítios de Ligação , COVID-19 , Simulação por Computador , Proteases 3C de Coronavírus , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Descoberta de Drogas/métodos , Simulação de Acoplamento Molecular , Pandemias , Inibidores de Proteases/química , Ligação Proteica , Bibliotecas de Moléculas Pequenas/química , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismoRESUMO
Improved sampling methodologies, more accurate force fields, and access to longer simulation time scales have led to an increased application of Relative Binding Free Energy (RBFE) calculations in drug discovery projects. In order to assess the strengths and limitations of such tools, adequate benchmark sets are required that challenge the methodology in certain well-defined aspects. We applied Free Energy Perturbation (FEP) calculations to six congeneric ligand pairs taken from the literature, in which addition of a functional group resulted in the displacement of buried binding site water molecules and compared the calculated relative binding free energies with the experimental ones. We started the perturbations from different initial solvation states and registered large inconsistencies (large hysteresis) between the calculated values. We furthermore applied a Grand Canonical Monte Carlo (GCMC) solvent sampling step prior to the FEP calculation that led to a smaller hysteresis for the simulations. By applying a hydration site analysis to the trajectories of the end-states of the perturbation, we could point out that the low accuracy of the predictions as well as the high dependence of the prediction on the chosen initial state is likely caused by the trapping of binding site water molecules and/or insufficient solvation of buried cavities that are formed upon completion of the perturbation. This work highlights that RBFE calculations can suffer from slow solvent exchange of buried parts of the binding sites with the bulk.
Assuntos
Água/química , Sítios de Ligação , Descoberta de Drogas , Ligantes , Modelos Moleculares , Conformação Molecular , Método de Monte Carlo , Solventes/química , Termodinâmica , Água/metabolismoRESUMO
Nuclear receptors (NRs) are ligand-inducible transcription factors that play an essential role in a multitude of physiological processes as well as diseases, rendering them attractive drug targets. Crystal structures revealed the binding site of NRs to be buried in the core of the protein, with no obvious route for ligands to access this cavity. The process of ligand binding is known to be an often-neglected contributor to the efficacy of drug candidates and is thought to influence the selectivity and specificity of NRs. While experimental methods generally fail to highlight the dynamic processes of ligand access or egress on the atomistic scale, computational methods have provided fundamental insight into the pathways connecting the buried binding pocket to the surrounding environment. Methods based on molecular dynamics (MD) and Monte Carlo simulations have been applied to identify pathways and quantify their capability to transport ligands. Here, we systematically review findings of more than 20 years of research in the field, including the applied methodology and controversies. Further, we establish a unified nomenclature to describe the pathways with respect to their location relative to protein secondary structure elements and summarize findings relevant to drug design. Lastly, we discuss the effect of NR interaction partners such as coactivators and corepressors, as well as mutations on the pathways.
Assuntos
Simulação por Computador , Receptores Citoplasmáticos e Nucleares/química , Ligantes , Modelos Químicos , Modelos Moleculares , Conformação ProteicaRESUMO
To identify new potential therapeutic targets for neurodegenerative diseases, we initiated activity-based protein profiling studies with withanolide A (WitA), a known neuritogenic constituent of Withania somnifera root with unknown mechanism of action. Molecular probes were designed and synthesized, and led to the discovery of the glucocorticoid receptor (GR) as potential target. Molecular modeling calculations using the VirtualToxLab predicted a weak binding affinity of WitA for GR. Neurite outgrowth experiments in human neuroblastoma SH-SY5Y cells further supported a glucocorticoid-dependent mechanism, finding that WitA was able to reverse the outgrowth inhibition mediated by dexamethasone (Dex). However, further GR binding and transactivation assays found no direct interference of WitA. Further molecular modeling analysis suggested that WitA, although forming several contacts with residues in the GR binding pocket, is lacking key stabilizing interactions as observed for Dex. Taken together, the data suggest that WitA-dependent induction of neurite outgrowth is not through a direct effect on GR, but might be mediated through a closely related pathway. Further experiments should evaluate a possible role of GR modulators and/or related signaling pathways such as ERK, Akt, NF-κB, TRα, or Hsp90 as potential targets in the WitA-mediated neuromodulatory effects.
Assuntos
Receptores de Glucocorticoides/metabolismo , Vitanolídeos/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Dexametasona/química , Dexametasona/metabolismo , Dexametasona/farmacologia , Glucocorticoides/química , Glucocorticoides/metabolismo , Glucocorticoides/farmacologia , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Simulação de Acoplamento Molecular , NF-kappa B/metabolismo , Neuritos/efeitos dos fármacos , Neuritos/metabolismo , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Ligação Proteica , Estrutura Terciária de Proteína , Receptores de Glucocorticoides/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Vitanolídeos/farmacologia , Vitanolídeos/uso terapêuticoRESUMO
A library of extracts from plants used in Chinese Traditional Medicine was screened for inhibition of T lymphocyte proliferation. An ethyl acetate extract from aerial parts of Artemisia argyi showed promising activity and was submitted to HPLC-based activity profiling to track the active compounds. From the most active time window, three guaianolides (1, 2, and 5) and two seco-tanapartholides (3 and 4) were identified and, in a less active time window, five new sesquiterpene lactones (8-11, 17), along with six known sesquiterpene lactones and two known flavonoids. The absolute configurations of compounds 1, 2, 5-10, 13-15, 17, and 18 were established by comparison of experimental with calculated electronic circular dichroism (ECD) spectra. For seco-tanapartholides B (3) and A (4), ECD yielded ambiguous results, and their absolute configurations were determined by comparing experimental and calculated vibrational circular dichroism (VCD) spectra. Compounds 1-5 showed significant, noncytotoxic inhibition of T lymphocyte proliferation, with IC50 values between 1.0 and 3.7 µM.