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1.
J Med Chem ; 2019 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-31762274

RESUMO

There is increasing evidence that histone deacetylase (HDAC) inhibitors can (re)sensitize cancer cells for chemotherapeutics via 'epigenetic priming'. In this work, we describe the synthesis of a series of class I selective HDAC inhibitors with 2-aminoanilides as zinc-binding groups. Several of the synthesized compounds revealed potent inhibition of the class I HDAC isoforms HDAC1, 2 and/or 3 and promising antiproliferative effects in the human ovarian cancer cell line A2780 and the human squamous carcinoma cell line Cal27. Selected compounds were investigated in a cellular model of platinum resistance. In particular compound 2a revealed potent chemosensitizing properties and full reversal of cisplatin resistance in Cal27CisR cells. This effect is related to a synergistic increase in caspase 3/7 activation and induction of apoptosis. Thus, this work demonstrates that pan-HDAC inhibition or dual class I/class IIb inhibition is not required for full reversal of cisplatin resistance.

2.
J Comput Chem ; 2019 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-31618459

RESUMO

Lipases are essential and widely used biocatalysts. Hence, the production of lipases requires a detailed understanding of the molecular mechanism of its folding and secretion. Lipase A from Pseudomonas aeruginosa, PaLipA, constitutes a prominent example that has additional relevance because of its role as a virulence factor in many diseases. PaLipA requires the assistance of a membrane-integrated steric chaperone, the lipase-specific foldase Lif, to achieve its enzymatically active state. However, the molecular mechanism of how Lif activates its cognate lipase has remained elusive. Here, we show by molecular dynamics simulations at the atomistic level and potential of mean force computations that Lif catalyzes the activation process of PaLipA by structurally stabilizing an intermediate PaLipA conformation, particularly a ß-sheet in the region of residues 17-30, such that the opening of PaLipA's lid domain is facilitated. This opening allows substrate access to PaLipA's catalytic site. A surprising and so far not fully understood aspect of our study is that the open state of PaLipA is unstable compared to the closed one according to our computational and in vitro biochemical results. We thus speculate that further interactions of PaLipA with the Xcp secretion machinery and/or components of the extracellular matrix contribute to the remaining activity of secreted PaLipA. © 2019 Wiley Periodicals, Inc.

3.
J Biol Chem ; 294(47): 17978-17987, 2019 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-31615893

RESUMO

cAMP acts as a second messenger in many cellular processes. Three protein types mainly mediate cAMP-induced effects: PKA, exchange protein directly activated by cAMP (Epac), and cyclic nucleotide-modulated channels (cyclic nucleotide-gated or hyperpolarization-activated and cyclic nucleotide-modulated (HCN) channels). Discrimination among these cAMP signaling pathways requires specific targeting of only one protein. Previously, cAMP modifications at position N 6 of the adenine ring (PKA) and position 2'-OH of the ribose (Epac) have been used to produce target-selective compounds. However, cyclic nucleotide-modulated ion channels were usually outside of the scope of these previous studies. These channels are widely distributed, so possible channel cross-activation by PKA- or Epac-selective agonists warrants serious consideration. Here we demonstrate the agonistic effects of three PKA-selective cAMP derivatives, N 6-phenyladenosine-3',5'-cyclic monophosphate (N 6-Phe-cAMP), N 6-benzyladenosine-3',5'-cyclic monophosphate (N 6-Bn-cAMP), and N 6-benzoyl-adenosine-3',5'-cyclic monophosphate (N 6-Bnz-cAMP), on murine HCN2 pacemaker channels. Electrophysiological characterization in Xenopus oocytes revealed that these derivatives differ in apparent affinities depending on the modification type but that their efficacy and effects on HCN2 activation kinetics are similar to those of cAMP. Docking experiments suggested a pivotal role of Arg-635 at the entrance of the binding pocket in HCN2, either causing stabilizing cation-π interactions with the aromatic ring in N 6-Phe-cAMP or N 6-Bn-cAMP or a steric clash with the aromatic ring in N 6-Bnz-cAMP. A reduced apparent affinity of N 6-Phe-cAMP toward the variants R635A and R635E strengthened that notion. We conclude that some PKA activators also effectively activate HCN2 channels. Hence, when studying PKA-mediated cAMP signaling with cAMP derivatives in a native environment, activation of HCN channels should be considered.

4.
ACS Chem Biol ; 2019 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-31638770

RESUMO

Drug optimization is guided by biophysical methods with increasing popularity. In the context of lead structure modifications, the introduction of methyl groups is a simple but potentially powerful approach. Hence, it is crucial to systematically investigate the influence of ligand methylation on biophysical characteristics such as thermodynamics. Here, we investigate the influence of ligand methylation in different positions and combinations on the drug-kinase interaction. Binding modes and complex structures were analyzed using protein crystallography. Thermodynamic signatures were measured via isothermal titration calorimetry (ITC). An extensive computational analysis supported the understanding of the underlying mechanisms. We found that not only position but also stereochemistry of the methyl group has an influence on binding potency as well as the thermodynamic signature of ligand binding to the protein. Strikingly, the combination of single methyl groups does not lead to additive effects. In our case, the merger of two methyl groups in one ligand leads to an entirely new alternative ligand binding mode in the protein ligand complex. Moreover, the combination of the two methyl groups also resulted in a nonadditive thermodynamic profile of ligand binding. Molecular dynamics (MD) simulations revealed distinguished characteristic motions of the ligands in solution explaining the pronounced thermodynamic changes. The unexpected drastic change in protein ligand interaction highlights the importance of crystallographic control even for minor modifications such as the introduction of a methyl group. For an in-depth understanding of ligand binding behavior, MD simulations have shown to be a powerful tool.

5.
Bioorg Med Chem ; 27(20): 115079, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31500943

RESUMO

Lantibiotics are antimicrobial peptides produced by Gram-positive bacteria and active in the nanomolar range. Nisin is the most intensely studied and used lantibiotic, with applications as food preservative and recognized potential for clinical usage. However, different bacteria that are pathogenic for humans and do not produce nisin, including Streptococcus agalactiae, show an innate resistance that has been related to the nisin resistance protein (NSR), a membrane-associated protease. Here, we report the first-in-class small-molecule inhibitors of SaNSR identified by virtual screening based on a previously derived structural model of the nisin/NSR complex. The inhibitors belong to three different chemotypes, of which the halogenated phenyl-urea derivative NPG9 is the most potent one. Co-administration of NPG9 with nisin yields increased potency compared to nisin alone in SaNSR-expressing bacteria. The binding mode of NPG9, predicted with molecular docking and validated by extensive molecular dynamics simulations, confirms a structure-activity relationship derived from the in vivo data. Saturation transfer difference-NMR experiments demonstrate direct binding of NPG9 to SaNSR and agree with the predicted binding mode. Our results demonstrate the potential to overcome SaNSR-related lantibiotic resistance by small molecules.

6.
Chemistry ; 25(64): 14613-14624, 2019 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-31498478

RESUMO

Histamine H4 receptor (H4 R) orthologues are G-protein-coupled receptors (GPCRs) that exhibit species-dependent basal activity. In contrast to the basally inactive mouse H4 R (mH4 R), human H4 R (hH4 R) shows a high degree of basal activity. We have performed long-timescale molecular dynamics simulations and rigidity analyses on wild-type hH4 R, the experimentally characterized hH4 R variants S179M, F169V, F169V+S179M, F168A, and on mH4 R to investigate the molecular nature of the differential basal activity. H4 R variant-dependent differences between essential motifs of GPCR activation and structural stabilities correlate with experimentally determined basal activities and provide a molecular explanation for the differences in basal activation. Strikingly, during the MD simulations, F16945.55 dips into the orthosteric binding pocket only in the case of hH4 R, thus adopting the role of an agonist and contributing to the stabilization of the active state. The results shed new light on the molecular mechanism of basal H4 R activation that are of importance for other GPCRs.


Assuntos
Fenilalanina/análogos & derivados , Receptores Histamínicos H4/agonistas , Animais , Sítios de Ligação , Domínio Catalítico , Humanos , Camundongos , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Fenilalanina/química , Estabilidade Proteica , Receptores Histamínicos H4/genética , Receptores Histamínicos H4/metabolismo
7.
Bioorg Med Chem ; 27(21): 115080, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31519378

RESUMO

Cells constantly need to adopt to changing environmental conditions, maintaining homeostasis and proteostasis. Heat shock proteins are a diverse class of molecular chaperones that assist proteins in folding to prevent stress-induced misfolding and aggregation. The heat shock protein of 90 kDa (HSP90) is the most abundant heat shock protein. While basal expression of HSP90 is essential for cell survival, in many tumors elevated HSP90 levels are found, which is often associated with bad prognosis. Therefore, HSP90 has emerged as a major target in tumor therapy. The HSP90 machinery is very complex in that it involves large conformational changes during the chaperoning cycle and a variety of co-chaperones. At the same time, this complexity offers a plethora of possibilities to interfere with HSP90 function. The best characterized class of HSP90 modulators are competitive inhibitors targeting the N-terminal ATP-binding pocket. Nineteen compounds of this class entered clinical trials. However, due to severe adverse effects, including induction of the heat shock response, no N-terminal inhibitor has been approved by the FDA so far. As alternatives, compounds commonly referred to as "C-terminal inhibitors" have been developed, either as natural product-based analogues or by rational design, which employ multiple mechanisms to modulate HSP90 function, including modulation of the interaction with co-chaperones, induction of conformational changes that influence the chaperoning cycle, or inhibition of C-terminal dimerization. In this review, we summarize the current development state of characteristic C-terminal inhibitors, with an emphasis on their (proposed) molecular modes of action and binding sites.

8.
Bioorg Med Chem ; 27(20): 115005, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31466836

RESUMO

Five new metabolites, including the xanthone derivative wentixanthone A (1), the benzophenone wentiphenone A (2), the diastereomeric mixtures of the bianthrones wentibianthrone A (3a, b) and wentibianthrone B (4a, b), as well as (10R,10'S)-wentibianthrone C (5a) and (10R,10'R)-wentibianthrone C (5b) were obtained from the fungus Aspergillus wentii, isolated from soil of the hypersaline lake El Hamra in Wadi El-Natrun, Egypt. The structures of the isolated compounds were established by one and two-dimensional NMR and MS spectroscopic analysis. The relative configuration of bianthrones (3-5) was elucidated by comparison of experimental and computed 1H NMR chemical shifts. Results of biological assays are reported.

9.
Plant Cell ; 31(10): 2525-2539, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31363039

RESUMO

Evolution of the C4 photosynthetic pathway involved in some cases recruitment of housekeeping proteins through gene duplication and their further neofunctionalization. NADP-malic enzyme (ME), the most widespread C4 decarboxylase, has increased its catalytic efficiency and acquired regulatory properties that allowed it to participate in the C4 pathway. Here, we show that regulation of maize (Zea mays) C4-NADP-ME activity is much more elaborate than previously thought. Using mass spectrometry, we identified phosphorylation of the Ser419 residue of C4-NADP-ME in protein extracts of maize leaves. The phosphorylation event increases in the light, with a peak at Zeitgeber time 2. Phosphorylation of ZmC4-NADP-ME drastically decreases its activity as shown by the low residual activity of the recombinant phosphomimetic mutant. Analysis of the crystal structure of C4-NADP-ME indicated that Ser419 is involved in the binding of NADP at the active site. Molecular dynamics simulations and effective binding energy computations indicate a less favorable binding of the cofactor NADP in the phosphomimetic and the phosphorylated variants. We propose that phosphorylation of ZmC4-NADP-ME at Ser419 during the first hours in the light is a cellular mechanism that fine tunes the enzymatic activity to coordinate the carbon concentration mechanism with the CO2 fixation rate, probably to avoid CO2 leakiness from bundle sheath cells.

10.
EMBO Rep ; 20(10): e48191, 2019 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-31379073

RESUMO

The Sec translocon provides the lipid bilayer entry for ribosome-bound nascent chains and thus facilitates membrane protein biogenesis. Despite the appreciated role of the native environment in the translocon:ribosome assembly, structural information on the complex in the lipid membrane is scarce. Here, we present a cryo-electron microscopy-based structure of bacterial translocon SecYEG in lipid nanodiscs and elucidate an early intermediate state upon insertion of the FtsQ anchor domain. Insertion of the short nascent chain causes initial displacements within the lateral gate of the translocon, where α-helices 2b, 7, and 8 tilt within the membrane core to "unzip" the gate at the cytoplasmic side. Molecular dynamics simulations demonstrate that the conformational change is reversed in the absence of the ribosome, and suggest that the accessory α-helices of SecE subunit modulate the lateral gate conformation. Site-specific cross-linking validates that the FtsQ nascent chain passes the lateral gate upon insertion. The structure and the biochemical data suggest that the partially inserted nascent chain remains highly flexible until it acquires the transmembrane topology.

11.
Bioorg Med Chem ; 27(19): 115039, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31420257

RESUMO

Fluorescent tagging of bioactive molecules is a powerful tool to study cellular uptake kinetics and is considered as an attractive alternative to radioligands. In this study, we developed fluorescent histone deacetylase (HDAC) inhibitors and investigated their biological activity and cellular uptake kinetics. Our approach was to introduce a dansyl group as a fluorophore in the solvent-exposed cap region of the HDAC inhibitor pharmacophore model. Three novel fluorescent HDAC inhibitors were synthesized utilizing efficient submonomer protocols followed by the introduction of a hydroxamic acid or 2-aminoanilide moiety as zinc-binding group. All compounds were tested for their inhibition of selected HDAC isoforms, and docking studies were subsequently performed to rationalize the observed selectivity profiles. All HDAC inhibitors were further screened in proliferation assays in the esophageal adenocarcinoma cell lines OE33 and OE19. Compound 2, 6-((N-(2-(benzylamino)-2-oxoethyl)-5-(dimethylamino)naphthalene)-1-sulfonamido)-N-hydroxyhexanamide, displayed the highest HDAC inhibitory capacity as well as the strongest anti-proliferative activity. Fluorescence microscopy studies revealed that compound 2 showed the fastest uptake kinetic and reached the highest absolute fluorescence intensity of all compounds. Hence, the rapid and increased cellular uptake of 2 might contribute to its potent anti-proliferative properties.

12.
Bioorg Med Chem ; 27(19): 115044, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31443950

RESUMO

Platinum compounds are the first-line therapy for many types of cancer. However, drug resistance has frequently been reported for and is a major limitation of platinum-based chemotherapy in the clinic. In the current study, we examined the anti-tumor activity of phomoxanthone A (PXA), a tetrahydroxanthone dimer isolated from the endophytic fungus Phomopsis longicolla, in several solid cancer cell lines and their cisplatin-resistant sub-cell lines. PXA showed strong cytotoxic effects with IC50 values in the high nanomolar or low micromolar range in MTT assays. IC50 values of PXA were lower than those of cisplatin. Remarkably, equipotent anti-cancer activity was found in cisplatin-sensitive and respective cisplatin-resistant cells. Anticancer effects of PXA were studied in further detail in ovarian cancer (A2780) and bladder cancer (J82) cell pairs. PXA led to rapid depolarization of the mitochondrial membrane potential and strong activation of caspase 3 and 7, eventually resulting in strong induction of apoptosis. These effects occurred again both in sensitive and resistant cell lines. IC50 values of PXA from MTT and mitochondrial membrane depolarization assays were in good agreement. Configurational free energy computations indicate that both the neutral and singly negatively charged PXA show membrane partitioning and can penetrate the inner mitochondrial membrane. PXA treatment did not damage the plasma membranes of cancer cells, thus excluding unspecific membrane effects. Further, PXA had neither an effect on intracellular ROS nor on reduction of ROS after hydrogen peroxide treatment. In conclusion, our studies present PXA as a natural compound with strong apoptotic anticancer effects against platinum-resistant solid cancers. This may open new treatment options in clinically resistant malignancies.

13.
FASEB J ; 33(10): 11507-11527, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31345061

RESUMO

We previously reported that inactivation of the transmembrane taurine transporter (TauT or solute carrier 6a6) causes early retinal degeneration in mice. Compatible with taurine's indispensability for cell volume homeostasis, protein stabilization, cytoprotection, antioxidation, and immuno- and neuromodulation, mice develop multisystemic dysfunctions (hearing loss; liver fibrosis; and behavioral, heart, and skeletal muscle abnormalities) later on. Here, by genetic, cell biologic, in vivo 1H-magnetic resonance spectroscopy and molecular dynamics simulation studies, we conducted in-depth characterization of a novel disorder: human TAUT deficiency. Loss of TAUT function due to a homozygous missense mutation caused panretinal degeneration in 2 brothers. TAUTp.A78E still localized in the plasma membrane but is predicted to impact structural stabilization. 3H-taurine uptake by peripheral blood mononuclear cells was reduced by 95%, and taurine levels were severely reduced in plasma, skeletal muscle, and brain. Extraocular dysfunctions were not yet detected, but significantly increased urinary excretion of 8-oxo-7,8-dihydroguanosine indicated generally enhanced (yet clinically unapparent) oxidative stress and RNA oxidation, warranting continuous broad surveillance.-Preising, M. N., Görg, B., Friedburg, C., Qvartskhava, N., Budde, B. S., Bonus, M., Toliat, M. R., Pfleger, C., Altmüller, J., Herebian, D., Beyer, M., Zöllner, H. J., Wittsack, H.-J., Schaper, J., Klee, D., Zechner, U., Nürnberg, P., Schipper, J., Schnitzler, A., Gohlke, H., Lorenz, B., Häussinger, D., Bolz, H. J. Biallelic mutation of human SLC6A6 encoding the taurine transporter TAUT is linked to early retinal degeneration.

14.
Sci Rep ; 9(1): 10068, 2019 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-31296930

RESUMO

The N-methyl-D-aspartate subfamily of ionotropic glutamate receptors (NMDARs) is well known for its important roles in the central nervous system (CNS), e.g. learning and memory formation. Besides the CNS, NMDARs are also expressed in numerous peripheral tissues including the pancreas, kidney, stomach, and blood cells, where an understanding of their physiological and pathophysiological roles is only evolving. Whereas subunit composition increases functional diversity of NMDARs, a great number of endogenous cues tune receptor signaling. Here, we characterized the effects of the steroid bile salts cholate and chenodeoxycholate (CDC) on recombinantly expressed NMDARs of defined molecular composition. CDC inhibited NMDARs in an isoform-dependent manner, preferring GluN2D and GluN3B over GluN2A and GluN2B receptors. Determined IC50 values were in the range of bile salt serum concentrations in severe cholestatic disease states, pointing at a putative pathophysiological significance of the identified receptor modulation. Both pharmacological and molecular simulation analyses indicate that CDC acts allosterically on GluN2D, whereas it competes with agonist binding on GluN3B receptors. Such differential modes of inhibition may allow isoform-specific targeted interference with the NMDAR/bile salt interaction. In summary, our study provides further molecular insight into the modulation of NMDARs by endogenous steroids and points at a putative pathophysiological role of the receptors in cholestatic disease.

15.
Sci Rep ; 9(1): 8869, 2019 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-31222090

RESUMO

The structure, mechanism of action and copper stoichiometry of the transmembrane sensor domain of the plant ethylene receptor ETR1 and homologs have remained elusive, hampering the understanding on how the perception of the plant hormone ethylene is transformed into a downstream signal. We generated the first structural model of the transmembrane sensor domain of ETR1 by integrating ab initio structure prediction and coevolutionary information. To refine and independently validate the model, we determined protein-related copper stoichiometries on purified receptor preparations and explored the helix arrangement by tryptophan scanning mutagenesis. All-atom molecular dynamics simulations of the dimeric model reveal how ethylene can bind proximal to the copper ions in the receptor, illustrating the initial stages of the ethylene perception process.

16.
Biophys J ; 116(12): 2411-2422, 2019 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-31130235

RESUMO

A highly specific molecular interaction of diffusible ligands with their receptors belongs to the key processes in cellular signaling. Because an appropriate method to monitor the unitary binding events is still missing, most of our present knowledge is based on ensemble signals recorded from a big number of receptors, such as ion currents or fluorescence changes of suitably labeled receptors, and reasoning from these data to the ligand binding. To study the binding process itself, appropriately tagged ligands are required that fully activate the receptors and report the binding at the same time. Herein, we tailored a series of 18 novel fluorescent cyclic nucleotide derivatives by attaching 6 different dyes via different alkyl linkers to the 8-position of the purine ring of cGMP or cAMP. The biological activity was determined in inside-out macropatches containing either homotetrameric (CNGA2), heterotetrameric (CNGA2:CNGA4:CNGB1b), or hyperpolarization-activated cyclic nucleotide-modulated (HCN2) channels. All these novel fluorescent ligands are efficient to activate the channels, and the potency of most of them significantly exceeded that of the natural cyclic nucleotides cGMP or cAMP. Moreover, some of them showed an enhanced brightness when bound to the channels. The best of our derivatives bear great potential to systematically analyze the activation mechanism in CNG and HCN channels, at both the level of ensemble and single-molecule analyses.

17.
Plant Physiol ; 180(3): 1564-1581, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31040174

RESUMO

Iron is a key transition element in the biosphere and is crucial for living organisms, although its cellular excess can be deleterious. Maintaining the balance of optimal iron availability in the model plant Arabidopsis (Arabidopsis thaliana) requires the precise operation of iron import through the principal iron transporter IRON-REGULATED TRANSPORTER1 (IRT1). Targeted inhibition of IRT1 can prevent oxidative stress, thus promoting plant survival. Here, we report the identification of an IRT1 inhibitor, namely the C2 domain-containing peripheral membrane protein ENHANCED BENDING1 (EHB1). EHB1 interacts with the cytoplasmically exposed variable region of IRT1, and we demonstrate that this interaction is greatly promoted by the presence of calcium. We found that EHB1 binds lipids characteristic of the plasma membrane, and the interaction between EHB1 and plant membranes is calcium-dependent. Molecular simulations showed that EHB1 membrane binding is a two-step process that precedes the interaction between EHB1 and IRT1. Genetic and physiological analyses indicated that EHB1 acts as a negative regulator of iron acquisition. The presence of EHB1 prevented the IRT1-mediated complementation of iron-deficient fet3fet4 yeast (Saccharomyces cerevisiae). Our data suggest that EHB1 acts as a direct inhibitor of IRT1-mediated iron import into the cell. These findings represent a major step in understanding plant iron acquisition, a process that underlies the primary production of bioavailable iron for land ecosystems.

18.
J Chem Inf Model ; 59(6): 2522-2528, 2019 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-31120747

RESUMO

We present PACKMOL-Memgen, a simple-to-use, generalized workflow for automated building of membrane-protein-lipid-bilayer systems based on open-source tools including Packmol, memembed, pdbremix, and AmberTools. Compared with web-interface-based related tools, PACKMOL-Memgen allows setup of multiple configurations of a system in a user-friendly and efficient manner within minutes. The generated systems are well-packed and thus well-suited as starting configurations in MD simulations under periodic boundary conditions, requiring only moderate equilibration times. PACKMOL-Memgen is distributed with AmberTools and runs on most computing platforms, and its output can also be used for CHARMM or adapted to other molecular-simulation packages.

19.
J Chem Theory Comput ; 15(5): 3331-3343, 2019 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-30998331

RESUMO

Modulating protein activity with small-molecules binding to cryptic pockets offers great opportunities to overcome hurdles in drug design. Cryptic sites are atypical binding sites in proteins that are closed in the absence of a stabilizing ligand and are thus inherently difficult to identify. Many studies have proposed methods to predict cryptic sites. However, a general approach to prospectively sample open conformations of these sites and to identify cryptic pockets in an unbiased manner suitable for structure-based drug design remains elusive. Here, we describe an all-atom, explicit cosolvent, molecular dynamics (MD) simulations-based workflow to sample the open states of cryptic sites and identify opened pockets, in a manner that does not require a priori knowledge about these sites. Furthermore, the workflow relies on a target-independent parametrization that only distinguishes between binding pockets for peptides or small molecules. We validated our approach on a diverse test set of seven proteins with crystallographically determined cryptic sites. The known cryptic sites were found among the three highest-ranked predicted cryptic sites, and an open site conformation was sampled and selected for most of the systems. Crystallographic ligand poses were well reproduced by docking into these identified open conformations for five of the systems. When the fully open state could not be reproduced, we were still able to predict the location of the cryptic site, or identify other cryptic sites that could be retrospectively validated with knowledge of the protein target. These characteristics render our approach valuable for investigating novel protein targets without any prior information.


Assuntos
Desenho de Drogas , Simulação de Dinâmica Molecular , Proteínas/química , Ligantes , Estrutura Molecular
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