RESUMEN
Human insulin-like peptide 5 (INSL5) is a gut hormone produced by colonic L-cells, and its biological functions are mediated by Relaxin Family Peptide Receptor 4 (RXFP4). Our preliminary data indicated that RXFP4 agonists are potential drug leads for the treatment of constipation. More recently, we designed and developed a novel RXFP4 antagonist, A13-nR that was shown to block agonist-induced activity in cells and animal models. We showed that A13-nR was able to block agonist-induced increases in colon motility in mice of both genders that express the receptor, RXFP4. Our data also showed that colorectal propulsion induced by intracolonic administration of short-chain fatty acids was antagonized by A13-nR. Therefore, A13-nR is an important research tool and potential drug lead for the treatment of colon motility disorders, such as bacterial diarrhea. However, A13-nR acted as a partial agonist at high concentrations in vitro and demonstrated modest antagonist potency (â¼35 nM). Consequently, the primary objective of this study is to pinpoint novel modifications to A13-nR that eliminate partial agonist effects while preserving or augmenting antagonist potency. In this work, we detail the creation of a series of A13-nR-modified analogues, among which analogues 3, 4, and 6 demonstrated significantly improved RXFP4 affinity (â¼3 nM) with reduced partial agonist activity, enhanced antagonist potency (â¼10 nM) and maximum agonist inhibition (â¼80 %) when compared with A13-nR. These compounds have potential as candidates for further preclinical evaluations, marking a significant stride toward innovative therapeutics for colon motility disorders.
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Insulina , Receptores Acoplados a Proteínas G , Receptores de Péptidos , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Animales , Humanos , Ratones , Masculino , Receptores de Péptidos/metabolismo , Receptores de Péptidos/antagonistas & inhibidores , Receptores de Péptidos/agonistas , Insulina/metabolismo , Femenino , Motilidad Gastrointestinal/efectos de los fármacos , Células HEK293 , Ratones Endogámicos C57BL , ProteínasRESUMEN
Clioquinol (5-chloro-7-iodo-8-hydroxyquinoline) is an antimicrobial agent whose actions as a zinc or copper ionophore and an iron chelator revived the interest in similar compounds for the treatment of fungal and bacterial infections, neurodegeneration and cancer. Recently, we reported zinc ionophores, including clioquinol, cause vasorelaxation in isolated arteries through mechanisms that involve sensory nerves, endothelium and vascular smooth muscle. Here, we report that clioquinol also uniquely acts as a competitive alpha-1 (α1) adrenoceptor antagonist. We employed ex vivo functional vascular contraction and pharmacological techniques in rat isolated mesenteric arteries, receptor binding assays using stabilized solubilized α1 receptor variants, or wild-type human α1-adrenoceptors transfected in COS-7 cells (African green monkey kidney fibroblast-like cells), and molecular dynamics homology modelling based on the recently published α1A adrenoceptor cryo-EM and α1B crystal structures. At higher concentrations, all ionophores including clioquinol cause a non-competitive antagonism of agonist-mediated contraction due to intracellular zinc delivery, as reported previously. However, at lower concentration ranges, clioquinol has an additional mechanism of competitively inhibiting α1-adrenoceptors that contributes to decreasing vascular contractility. Molecular dynamic simulation showed that clioquinol binds stably to the orthosteric binding site (Asp106) of the receptor, confirming the structural basis for competitive α1-adrenoceptor antagonism by clioquinol.
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Clioquinol , Ratas , Humanos , Animales , Chlorocebus aethiops , Clioquinol/farmacología , Oxiquinolina , Receptores Adrenérgicos alfa 1/metabolismo , Ionóforos , ZincRESUMEN
α1A-, α1B-, and α1D-adrenoceptors (α1-ARs) are members of the adrenoceptor G protein-coupled receptor family that are activated by adrenaline (epinephrine) and noradrenaline. α1-ARs are clinically targeted using antagonists that have minimal subtype selectivity, such as prazosin and tamsulosin, to treat hypertension and benign prostatic hyperplasia, respectively. Abundant expression of α1-ARs in the heart and central nervous system (CNS) makes these receptors potential targets for the treatment of cardiovascular and CNS disorders, such as heart failure, epilepsy, and Alzheimer's disease. Our understanding of the precise physiological roles of α1-ARs, however, and their involvement in disease has been hindered by the lack of sufficiently subtype-selective tool compounds, especially for α1B-AR. Here, we report the discovery of 4-[(2-hydroxyethyl)amino]-6-methyl-2H-chromen-2-one (Cpd1), as an α1B-AR antagonist that has 10-15-fold selectivity over α1A-AR and α1D-AR. Through computational and site-directed mutagenesis studies, we have identified the binding site of Cpd1 in α1B-AR and propose the molecular basis of α1B-AR selectivity, where the nonconserved V19745.52 residue plays a major role, with contributions from L3146.55 within the α1B-AR pocket. By exploring the structure-activity relationships of Cpd1 at α1B-AR, we have also identified 3-[(cyclohexylamino)methyl]-6-methylquinolin-2(1H)-one (Cpd24), which has a stronger binding affinity than Cpd1, albeit with reduced selectivity for α1B-AR. Cpd1 and Cpd24 represent potential leads for α1B-AR-selective drug discovery and novel tool molecules to further study the physiology of α1-ARs.
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Prazosina , Receptores Adrenérgicos alfa 1 , Receptores Adrenérgicos alfa 1/metabolismo , Tamsulosina , NorepinefrinaRESUMEN
Lipid-based formulation (LBF) is an effective approach for delivering hydrophobic drugs into the systemic circulation by oral administration. However, much of the physical detail regarding the colloidal behavior of LBFs and their interactions with the contents of the gastrointestinal (GI) environment is not well characterized. Recently, researchers have started to use molecular dynamics (MD) simulations to investigate the colloidal behavior of LBF systems and their interactions with bile and other materials present in the GI tract. MD is a computational method, based on classical mechanics, that simulates the physical movements of atoms and provides atomic-scale information that cannot easily be retrieved using experimental investigations. MD can provide insight into assist the development of drug formulations in a cost and time-effective manner. This review summarizes the application of MD simulation to the study of bile, bile salts, and LBFs and their behavior within the GI environment and also discusses MD simulations of lipid-based mRNA vaccine formulations.
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Ácidos y Sales Biliares , Bilis , Bilis/química , Simulación de Dinámica Molecular , Composición de Medicamentos , Liposomas , Lípidos/química , SolubilidadRESUMEN
Peptide toxins that adopt the ShK fold can inhibit the voltage-gated potassium channel KV1.3 with IC50 values in the pM range and are therefore potential leads for drugs targeting autoimmune and neuroinflammatory diseases. Nuclear magnetic resonance (NMR) relaxation measurements and pressure-dependent NMR have shown that, despite being cross-linked by disulfide bonds, ShK itself is flexible in solution. This flexibility affects the local structure around the pharmacophore for the KV1.3 channel blockade and, in particular, the relative orientation of the key Lys and Tyr side chains (Lys22 and Tyr23 in ShK) and has implications for the design of KV1.3 inhibitors. In this study, we have performed molecular dynamics (MD) simulations on ShK and a close homologue, HmK, to probe the conformational space occupied by the Lys and Tyr residues, and docked the different conformations with a recently determined cryo-EM structure of the KV1.3 channel. Although ShK and HmK have 60% sequence identity, their dynamic behaviors are quite different, with ShK sampling a broad range of conformations over the course of a 5 µs MD simulation, while HmK is relatively rigid. We also investigated the importance of conformational dynamics, in particular the distance between the side chains of the key dyad Lys22 and Tyr23, for binding to KV1.3. Although these peptides have quite different dynamics, the dyad in both adopts a similar configuration upon binding, revealing a conformational selection upon binding to KV1.3 in the case of ShK. Both peptides bind to KV1.3 with Lys22 occupying the pore of the channel. Intriguingly, the more flexible peptide, ShK, binds with significantly higher affinity than HmK.
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Venenos de Cnidarios , Anémonas de Mar , Animales , Canal de Potasio Kv1.3/química , Canal de Potasio Kv1.3/metabolismo , Venenos de Cnidarios/química , Venenos de Cnidarios/metabolismo , Venenos de Cnidarios/farmacología , Anémonas de Mar/química , Anémonas de Mar/metabolismo , Péptidos/química , Conformación Molecular , Bloqueadores de los Canales de Potasio/farmacología , Bloqueadores de los Canales de Potasio/química , Canal de Potasio Kv.1.2/metabolismoRESUMEN
Human immunodeficiency virus type I (HIV-1) is a retrovirus that infects cells of the host's immune system leading to acquired immunodeficiency syndrome and potentially death. Although treatments are available to prevent its progression, HIV-1 remains a major burden on health resources worldwide. Continued emergence of drug-resistance mutations drives the need for novel drugs that can inhibit HIV-1 replication through new pathways. The viral protein reverse transcriptase (RT) plays a fundamental role in the HIV-1 replication cycle, and multiple approved medications target this enzyme. In this study, fragment-based drug discovery was used to optimize a previously identified hit fragment (compound B-1), which bound RT at a novel site. Three series of compounds were synthesized and evaluated for their HIV-1 RT binding and inhibition. These series were designed to investigate different vectors around the initial hit in an attempt to improve inhibitory activity against RT. Our results show that the 4-position of the core scaffold is important for binding of the fragment to RT, and a lead compound with a cyclopropyl substitution was selected and further investigated. Requirements for binding to the NNRTI-binding pocket (NNIBP) and a novel adjacent site were investigated, with lead compound 27-a minimal but efficient NNRTI-offering a starting site for the development of novel dual NNIBP-Adjacent site inhibitors.
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Síndrome de Inmunodeficiencia Adquirida , Fármacos Anti-VIH , VIH-1 , Humanos , Inhibidores de la Transcriptasa Inversa/química , Transcriptasa Inversa del VIH , Síndrome de Inmunodeficiencia Adquirida/tratamiento farmacológico , Fármacos Anti-VIH/farmacología , Fármacos Anti-VIH/uso terapéuticoRESUMEN
While a range of strategies exist to accomplish peptide macrocyclization, they are frequently limited by the need for orthogonal protection or provide little opportunity for structural diversification. We have evaluated an efficient macrocyclization method that employs nucleophilic aromatic substitution (SNAr) to create thioether macrocycles. This versatile macrocyclization, orthogonal to conventional peptide synthesis, can be performed in solution on unprotected peptidomimetics or on resin-bound peptides with side-chain protection in place. We show that the electron-withdrawing groups present in the products can be further utilized in subsequent orthogonal reactions to alter the peptide properties or to add prosthetic groups. The macrocyclization strategy was applied to the design of melanocortin ligands, generating a library of potent melanocortin agonists that exhibit distinct subtype selectivity.
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Péptidos Cíclicos , Péptidos , Péptidos Cíclicos/farmacología , Péptidos Cíclicos/química , Ciclización , Péptidos/química , Receptores de Melanocortina , Biblioteca de GenesRESUMEN
Human transthyretin (hTTR) can form amyloid deposits that accumulate in nerves and organs, disrupting cellular function. Molecules such as tafamidis that bind to and stabilize the TTR tetramer can reduce such amyloid formation. Here, we studied the interaction of VCP-6 (2-((3,5-dichlorophenyl)amino)benzoic acid) with hTTR. VCP-6 binds to hTTR with 5 times the affinity of the cognate ligand, thyroxine (T4). The structure of the hTTR:VCP-6 complex was determined by X-ray crystallography at 1.52 Šresolution. VCP-6 binds deeper in the binding channel than T4 with the 3',5'-dichlorophenyl ring binding in the 'forward' mode towards the channel centre. The dichlorophenyl ring lies along the 2-fold axis coincident with the channel centre, while the 2-carboxylatephenylamine ring of VCP-6 is symmetrically displaced from the 2-fold axis, allowing the 2-carboxylate group to form a tight intermolecular hydrogen bond with Nζ of Lys15 and an intramolecular hydrogen bond with the amine of VCP-6, stabilizing its conformation and explaining the greater affinity of VCP-6 compared to T4. This arrangement maintains optimal halogen bonding interactions in the binding sites, via chlorine atoms rather than iodine of the thyroid hormone, thereby explaining why the dichloro substitution pattern is a stronger binder than either the diiodo or dibromo analogues.
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Amiloidosis , Prealbúmina , Humanos , Prealbúmina/metabolismo , Conformación Proteica , Modelos Moleculares , Sitios de Unión , Cristalografía por Rayos X , Amiloide , Proteínas Amiloidogénicas , HalógenosRESUMEN
The ability to predict cell-permeable candidate molecules has great potential to assist drug discovery projects. Large molecules that lie beyond the Rule of Five (bRo5) are increasingly important as drug candidates and tool molecules for chemical biology. However, such large molecules usually do not cross cell membranes and cannot access intracellular targets or be developed as orally bioavailable drugs. Here, we describe a random forest (RF) machine learning model for the prediction of passive membrane permeation rates developed using a set of over 1000 bRo5 macrocyclic compounds. The model is based on easily calculated chemical features/descriptors as independent variables. Our random forest (RF) model substantially outperforms a multiple linear regression model based on the same features and achieves better performance metrics than previously reported models using the same underlying data. These features include: (1) polar surface area in water, (2) the octanol-water partitioning coefficient, (3) the number of hydrogen-bond donors, (4) the sum of the topological distances between nitrogen atoms, (5) the sum of the topological distances between nitrogen and oxygen atoms, and (6) the multiple molecular path count of order 2. The last three features represent molecular flexibility, the ability of the molecule to adopt different conformations in the aqueous and membrane interior phases, and the molecular "chameleonicity." Guided by the model, we propose design guidelines for membrane-permeating macrocycles. It is anticipated that this model will be useful in guiding the design of large, bioactive molecules for medicinal chemistry and chemical biology applications.
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Compuestos Macrocíclicos , Hidrógeno , Aprendizaje Automático , Nitrógeno , Octanoles , Oxígeno , AguaRESUMEN
The SPRY domain-containing SOCS box protein-2 (SPSB2) plays a critical role in the degradation of inducible nitric oxide synthase (iNOS) in macrophages. In this study, we have conjugated a peptide inhibitor of the iNOS-SPSB2 interaction with a cell-penetrating peptide (CPP) for delivery into macrophages, and confirmed its binding to SPSB2. We have assessed the uptake of a fluorophore-tagged analogue by RAW 264.7 and immortalised bone marrow derived macrophage (iBMDM) cell lines, and shown that the CPP-peptide conjugate enhanced NO production. The findings of this study will be useful in further refinement of CPP-peptide conjugates as leads in the development of new antibiotics that target the host innate immune response.
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Péptidos de Penetración Celular , Óxido Nítrico , Péptidos de Penetración Celular/farmacología , Macrófagos/metabolismo , Modelos Moleculares , Óxido Nítrico Sintasa de Tipo II/metabolismoRESUMEN
α-adrenergic receptors (αARs) are G protein-coupled receptors that regulate vital functions of the cardiovascular and nervous systems. The therapeutic potential of αARs, however, is largely unexploited and hampered by the scarcity of subtype-selective ligands. Moreover, several aminergic drugs either show off-target binding to αARs or fail to interact with the desired subtype. Here, we report the crystal structure of human α1BAR bound to the inverse agonist (+)-cyclazosin, enabled by the fusion to a DARPin crystallization chaperone. The α1BAR structure allows the identification of two unique secondary binding pockets. By structural comparison of α1BAR with α2ARs, and by constructing α1BAR-α2CAR chimeras, we identify residues 3.29 and 6.55 as key determinants of ligand selectivity. Our findings provide a basis for discovery of α1BAR-selective ligands and may guide the optimization of aminergic drugs to prevent off-target binding to αARs, or to elicit a selective interaction with the desired subtype.
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Cristalografía por Rayos X , Receptores Adrenérgicos alfa 1/química , Sitios de Unión , Células HEK293 , Humanos , Ligandos , Lípidos/química , Modelos Moleculares , Quinazolinas/química , Quinazolinas/metabolismo , Quinoxalinas/química , Quinoxalinas/metabolismo , Receptores Adrenérgicos alfa 2/químicaRESUMEN
DsbA enzymes catalyze oxidative folding of proteins that are secreted into the periplasm of Gram-negative bacteria, and they are indispensable for the virulence of human pathogens such as Vibrio cholerae and Escherichia coli. Therefore, targeting DsbA represents an attractive approach to control bacterial virulence. X-ray crystal structures reveal that DsbA enzymes share a similar fold, however, the hydrophobic groove adjacent to the active site, which is implicated in substrate binding, is shorter and flatter in the structure of V.â cholerae DsbA (VcDsbA) compared to E.â coli DsbA (EcDsbA). The flat and largely featureless nature of this hydrophobic groove is challenging for the development of small molecule inhibitors. Using fragment-based screening approaches, we have identified a novel small molecule, based on the benzimidazole scaffold, that binds to the hydrophobic groove of oxidized VcDsbA with a KD of 446±10â µM. The same benzimidazole compound has â¼8-fold selectivity for VcDsbA over EcDsbA and binds to oxidized EcDsbA, with KD >3.5â mM. We generated a model of the benzimidazole complex with VcDsbA using NMR data but were unable to determine the structure of the benzimidazole bound EcDsbA using either NMR or X-ray crystallography. Therefore, a structural basis for the observed selectivity is unclear. To better understand ligand binding to these two enzymes we crystallized each of them in complex with a known ligand, the bile salt sodium taurocholate. The crystal structures show that taurocholate adopts different binding poses in complex with VcDsbA and EcDsbA, and reveal the protein-ligand interactions that stabilize the different modes of binding. This work highlights the capacity of fragment-based drug discovery to identify inhibitors of challenging protein targets. In addition, it provides a starting point for development of more potent and specific VcDsbA inhibitors that act through a novel anti-virulence mechanism.
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Proteínas de Escherichia coli , Vibrio cholerae , Antibacterianos/química , Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Bencimidazoles , Cristalografía por Rayos X , Escherichia coli , Humanos , Ligandos , Proteína Disulfuro IsomerasasRESUMEN
Selective agonists for the human M1 and M4 muscarinic acetylcholine receptors (mAChRs) are attractive candidates for the treatment of cognitive disorders, such as Alzheimer's disease and schizophrenia. Past efforts to optimize a ligand for selective agonism at any one of the M1-M5 mAChR subtypes has proven to be a significant challenge. Recently, research efforts have demonstrated that hybrid ligands may offer a potential solution to the lack of selectivity at mAChRs. In an attempt to design M1 mAChR selective agonists by hybridizing an M1 mAChR selective positive allosteric modulator [1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid] and a potent agonist [(4-[(4,5-dihydro-3-isoxazolyl)oxy]-N,N,N-trimethyl-2-butyn-1-aminium iodide) (iperoxo)], we unexpectedly discovered that these ligands possessed noticeable M2/M4 mAChR selectivity. Evaluation of truncated derivatives of the hybrid ligands at the M1-M5 mAChR subtypes suggests that the allosteric pharmacophore of iperoxo-based mAChR hybrid ligands likely sterically disrupts the allosteric site of the mAChRs, attenuating the efficacy of M1/M3/M5 mAChR responses compared to M2/M4 mAChRs, resulting in a preference for the M2/M4 mAChRs. However, at certain intermediate linker lengths, the effects of this apparent disruption of the allosteric site are diminished, restoring nonselective agonism and suggesting a possible allosteric interaction which is favorable to efficacy at all M1-M5 mAChRs.
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Isoxazoles , Receptor Muscarínico M1 , Regulación Alostérica , Animales , Células CHO , Cricetinae , Humanos , Ligandos , Compuestos de Amonio CuaternarioRESUMEN
Type III lipid-based formulations (LBFs) combine poorly water-soluble drugs with oils, surfactants, and cosolvents to deliver the drugs into the systemic circulation. However, the solubility of the drug can be influenced by the colloidal phases formed in the gastrointestinal tract as the formulation is dispersed and makes contact with bile and other materials present within the GI tract. Thus, an understanding of the phase behavior of LBFs in the gut is critical for designing efficient LBFs. Molecular dynamics (MD) simulation is a powerful tool for the study of colloidal systems. In this study, we modeled the internal structures of five type III LBFs of loratadine containing poly(ethylene oxide) nonionic surfactants polysorbate 80 and polyoxyl hydrogenated castor oil (Kolliphor RH40) using long-timescale MD simulations (0.4-1.7 µs). We also conducted experimental investigations (dilution of formulations with water) including commercial Claritin liquid softgel capsules. The simulations show that LBFs form continuous phase, water-swollen reverse micelles, and bicontinuous and phase-separated systems at different dilutions, which correlate with the experimental observations. This study supports the use of MD simulation as a predictive tool to determine the fate of LBFs composed of medium-chain lipids, polyethylene oxide surfactants, and polymers.
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Lípidos/química , Loratadina/química , Tensoactivos/química , Composición de Medicamentos , Excipientes/química , Simulación de Dinámica Molecular , Polisorbatos/química , Agua/químicaRESUMEN
OBJECTIVE: Molecular dynamics (MD) simulations provide an in silico method to study the structure of lipid-based formulations (LBFs) and the incorporation of poorly water-soluble drugs within such formulations. In order to validate the ability of MD to effectively model the properties of LBFs, this work investigates the well-known cyclosporine A formulations, Sandimmune® and Neoral®. Sandimmune® exhibits poor dispersibility and its absorption from the gastrointestinal tract is enhanced when administered after food, whereas Neoral® disperses comparatively well and shows no food effect. METHODS: MD simulations were performed of both LBFs to investigate the differences observed in fasted and fed conditions. These conditions were also tested using an in vitro experimental model of dispersion and digestion. RESULTS: These MD simulations were able to show that the food effect observed for Sandimmune® can be explained by large changes in drug solubilization on addition of bile. In contrast, Neoral® is well dispersed in water or in simulated fasted conditions, and this dispersion is relatively unchanged on moving to fed conditions. These differences were confirmed using dispersion and digestion in vitro experimental model. CONCLUSIONS: The current data suggests that MD simulations are a potential method to model the fate of LBFs in the gastrointestinal tract, predict their dispersion and digestion, investigate behaviour of APIs within the formulations, and provide insights into the clinical performance of LBFs.
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Ciclosporina/química , Lípidos/química , Bilis/química , Química Farmacéutica/métodos , Digestión , Excipientes/química , Simulación de Dinámica Molecular , Solubilidad/efectos de los fármacos , Agua/químicaRESUMEN
Cyclosporins are natural or synthetic undecapeptides with a wide range of actual and potential pharmaceutical applications. Several members of the cyclosporin compound family have remarkably high passive membrane permeabilities that are not well-described by simple structural metrics. Here we review experimental studies of cyclosporin structure and permeability, including cyclosporin-metal complexes. We also discuss models for the conformation-dependent permeability of cyclosporins and similar compounds. Finally, we identify current knowledge gaps in the literature and provide recommendations regarding future avenues of exploration.
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Permeabilidad de la Membrana Celular , Membrana Celular/metabolismo , Ciclosporinas/metabolismo , Animales , Ciclosporinas/química , Humanos , Modelos Químicos , Conformación ProteicaRESUMEN
The malaria vaccine candidate merozoite surface protein 2 (MSP2) has shown promise in clinical trials and is in part responsible for a reduction in parasite densities. However, strain-specific reductions in parasitaemia suggested that polymorphic regions of MSP2 are immuno-dominant. One strategy to bypass the hurdle of strain-specificity is to bias the immune response towards the conserved regions. Two mouse monoclonal antibodies, 4D11 and 9H4, recognise the conserved C-terminal region of MSP2. Although they bind overlapping epitopes, 4D11 reacts more strongly with native MSP2, suggesting that its epitope is more accessible on the parasite surface. In this study, a structure-based vaccine design approach was applied to the intrinsically disordered antigen, MSP2, using a crystal structure of 4D11 Fv in complex with its minimal binding epitope. Molecular dynamics simulations and surface plasmon resonance informed the design of a series of constrained peptides that mimicked the 4D11-bound epitope structure. These peptides were conjugated to keyhole limpet hemocyanin and used to immunise mice, with high to moderate antibody titres being generated in all groups. The specificities of antibody responses revealed that a single point mutation can focus the antibody response towards a more favourable epitope. This structure-based approach to peptide vaccine design may be useful not only for MSP2-based malaria vaccines, but also for other intrinsically disordered antigens.
RESUMEN
Our understanding of the factors affecting the stability of cyclic d/l peptide (CP) nanotubes remains underdeveloped. In this work, we investigate the impact of side chain alignment, hydrophobicity and charge on CP nanotube stability through X-ray crystallography, NMR spectroscopy and molecular dynamics (MD) simulations. We characterise the distinct CP-CP alignments that can form and identify stable and unstable dimers by MD simulation. We measure H-bond half-lives of synthesised CPs by 1 H-D exchange experiments and find good correlation with predicted CP-CP stabilities. We find that hydrophobic amino acids improve CP dimer stability but experimentally reduce solubility. Charged amino acids either increase or decrease CP dimer stability depending on the relative orientation and composition of charged groups. X-ray crystal structures are solved for two CPs, revealing non-tubular folded conformations. Ultimately, this work will assist the educated design of stable tubular structures for potential applications in biomedicine.
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Nanotubos de Péptidos , Nanotubos , Cristalografía , Cristalografía por Rayos X , Espectroscopía de Resonancia Magnética , Simulación de Dinámica Molecular , Péptidos CíclicosRESUMEN
Drug absorption from lipid-based formulations (LBFs) in the gastrointestinal (GI) tract is the result of a series of processes, including formulation dispersion, interaction with biliary and pancreatic secretions, drug solubilisation and supersaturation, and finally intestinal permeability. Optimal formulation design is dependent on a good understanding of the limitations to, and drivers of, absorption, but for LBFs the complexity of these processes makes data interpretation complex. The current study has re-examined a previous in vitro digestion-in situ perfusion model to increase physiological relevance and has used this model to examine drug absorption from LBFs. The composition of rat bile and jejunal fluid was also characterised to identify in vivo-relevant conditions. Digestion was initiated using rat bile/pancreatic fluid and the formulation and digestive enzymes mixed immediately prior to entry into the jejunum (allowing dilution/digestion to occur at the absorptive site). These conditions were employed to study drug absorption from LBFs of high (fenofibrate, FFB) and low (saquinavir, SQV) permeability compounds. The impact of polymeric precipitation inhibitors (PPIs) was also evaluated. For FFB, supersaturation, initiated by formulation interaction with biliary/pancreatic fluids, appeared to drive absorption and the addition of the PPIs poly(glycidyl methacrylate) (PPGAE) and hydroxypropylmethyl cellulose (HPMC), reduced drug precipitation, increased FFB supersaturation and increased absorption from a Type IV LBF of FFB. For a Type IIIB LBF however, PPIs were ineffective at increasing absorption. The impact of PPIs on the absorption of a less permeable drug, SQV, was similarly evaluated and again drug absorption appeared to be related to the extent of supersaturation, although in this case PPI were unable to promote absorption. For both FFB and SQV, drug absorption patterns obtained with the in vitro digestion-in situ perfusion mode, correlated well with in vitro supersaturation data and in vivo drug exposure data from oral bioavailability studies. The data are consistent with a mode of drug absorption where rapid dilution of LBFs with biliary and pancreatic secretions at the absorptive site in the upper small intestine drives transient supersaturation, that supersaturation is a significant driver of drug absorption for both low and high permeability drugs, and that PPIs delay drug precipitation, enhance supersaturation and promote drug absorption in a drug and formulation specific manner.
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Fenofibrato , Preparaciones Farmacéuticas , Administración Oral , Animales , Absorción Intestinal , Lípidos , Permeabilidad , Ratas , Saquinavir , SolubilidadRESUMEN
HYPOTHESIS: Non-ionic surfactants containing polyethylene oxide (PEO) chains are widely used in drug formulations, cosmetics, paints, textiles and detergents. High quality molecular dynamics models for PEO surfactants can give us detailed, atomic-scale information about the behavior of surfactant/water mixtures. SIMULATIONS: We used two molecular dynamics force fields (FFs), 2016H66 and 53A6DBW, to model the simple non-ionic PEO surfactant, hexaoxyethylene dodecyl ether (C12E6). We investigated surfactant/water mixtures that span the phase diagram of starting from randomly distributed arrangements. In some cases, we also started with prebuilt, approximate models. The simulations results were compared with the experimentally observed phase behavior. FINDINGS: Overall, this study shows that the spontaneous self-assembly of PEO non-ionic surfactants into different colloidal structures can be accurately modeled with MD simulations using the 2016H66 FF although transitions to well-formed hexagonal phase are slow. Of the two FFs investigated, the 2016H66 FF better reproduces the experimental phase behavior across all regions of the C12E6/water phase diagram.