RESUMEN
The synthesis of a water-soluble, phosphine-pegylated iridium(I) catalyst and its application in hydrogen isotope exchange (HIE) reactions in buffer is reported. The longer polyethylene glycol side chains on the phosphine increased the water solubility independently from the pH. HIE reactions of polar substrates in protic solvents were studied. DFT calculations gave further insights into the catalytic processes. The scope and limitation of the pegylated catalyst was studied in HIE reactions of several complex compounds in borax buffer at pHâ 9 and the best conditions were applied in a tritium experiment with the drug telmisartan.
RESUMEN
Kinetic aqueous or buffer solubility is important parameter measuring suitability of compounds for high throughput assays in early drug discovery while thermodynamic solubility is reserved for later stages of drug discovery and development. Kinetic solubility is also considered to have low inter-laboratory reproducibility because of its sensitivity to protocol parameters [1]. Presumably, this is why little efforts have been put to build QSPR models for kinetic in comparison to thermodynamic aqueous solubility. Here, we investigate the reproducibility and modelability of kinetic solubility assays. We first analyzed the relationship between kinetic and thermodynamic solubility data, and then examined the consistency of data from different kinetic assays. In this contribution, we report differences between kinetic and thermodynamic solubility data that are consistent with those reported by others [1, 2] and good agreement between data from different kinetic solubility campaigns in contrast to general expectations. The latter is confirmed by achieving high performing QSPR models trained on merged kinetic solubility datasets. The poor performance of QSPR model trained on thermodynamic solubility when applied to kinetic solubility dataset reinforces the conclusion that kinetic and thermodynamic solubilities do not correlate: one cannot be used as an ersatz for the other. This encourages for building predictive models for kinetic solubility. The kinetic solubility QSPR model developed in this study is freely accessible through the Predictor web service of the Laboratory of Chemoinformatics (https://chematlas.chimie.unistra.fr/cgi-bin/predictor2.cgi).
Asunto(s)
Descubrimiento de Drogas , Ensayos Analíticos de Alto Rendimiento , Solubilidad , Reproducibilidad de los Resultados , Agua , Aprendizaje AutomáticoRESUMEN
Despite beneficial effects in acute heart failure, the full therapeutic potential of recombinant relaxin-2 has been hampered by its short half-life and the need for intravenous administration limiting its use to intensive care units. A multiparametric optimization of the relaxin B-chain led to the identification of single chain lipidated peptide agonists of RXFP1 like SA10SC-RLX with subcutaneous bioavailability and extended half-life. SA10SC-RLX has sub nanomolar activity on cells expressing human RXFP1 and molecular modeling associated with the study of different RXFP1 mutants was used to decipher the mechanism of SA10SC-RLX interaction with RXFP1. Telemetry was performed in rat where SA10SC-RLX was able to engage RXFP1 after subcutaneous administration without tachyphylaxis after repeated dosing. Renal blood flow was then used as a translational model to evaluate RXFP1 activation. SA10SC-RLX increased renal blood flow and decreased renal vascular resistance in rats as reported for relaxin in humans. In conclusion, SA10SC-RLX mimics relaxin activity in in vitro and in vivo models of acute RXFP1 engagement. SA10SC-RLX represents a new class of long-lasting RXFP1 agonist, suitable for once daily subcutaneous administration in patients and potentially paving the way to new treatments for chronic fibrotic and cardiovascular diseases.
Asunto(s)
Relaxina , Humanos , Animales , Ratas , Relaxina/farmacología , Semivida , Circulación Renal , Modelos Moleculares , Administración Intravenosa , Receptores de Péptidos/genética , Receptores Acoplados a Proteínas GRESUMEN
Proprotein convertase subtilisin/kexin type 9 (PCSK9), identified as a regulator of low-density lipoprotein receptor (LDLR), plays a major role in cardiovascular diseases (CVD). Recently, Pep2-8, a small peptide with discrete three-dimensional structure, was found to inhibit the PCSK9/LDLR interaction. In this paper, we describe the modification of this peptide using stapled peptide and SIP technologies. Their combination yielded potent compounds such as 18 that potently inhibited the binding of PCSK9 to LDLR (KD = 6 ± 1 nM) and restored in vitro LDL uptake by HepG2 cells in the presence of PCSK9 (EC50 = 175 ± 40 nM). The three-dimensional structures of key peptides were extensively studied by circular dichroism and nuclear magnetic resonance, and molecular dynamics simulations allowed us to compare their binding mode to tentatively rationalize structure-activity relationships (SAR).
Asunto(s)
Lisina/farmacología , Inhibidores de PCSK9 , Péptidos/farmacología , Inhibidores de Serina Proteinasa/farmacología , Relación Dosis-Respuesta a Droga , Células Hep G2 , Humanos , Lisina/química , Modelos Moleculares , Estructura Molecular , Péptidos/síntesis química , Péptidos/química , Proproteína Convertasa 9/metabolismo , Inhibidores de Serina Proteinasa/síntesis química , Inhibidores de Serina Proteinasa/química , Relación Estructura-ActividadRESUMEN
The insulin-like peptide human relaxin-2 was identified as a hormone that, among other biological functions, mediates the hemodynamic changes occurring during pregnancy. Recombinant relaxin-2 (serelaxin) has shown beneficial effects in acute heart failure, but its full therapeutic potential has been hampered by its short half-life and the need for intravenous administration limiting its use to intensive care units. In this study, we report the development of long-acting potent single-chain relaxin peptide mimetics. Modifications in the B-chain of relaxin, such as the introduction of specific mutations and the trimming of the sequence to an optimal size, resulted in potent, structurally simplified peptide agonists of the relaxin receptor Relaxin Family Peptide Receptor 1 (RXFP1) (e.g., 54). Introduction of suitable spacers and fatty acids led to the identification of single-chain lipidated peptide agonists of RXFP1, with sub-nanomolar activity, high subcutaneous bioavailability, extended half-lives, and in vivo efficacy (e.g., 64).
Asunto(s)
Lipopéptidos/farmacología , Receptores Acoplados a Proteínas G/agonistas , Receptores de Péptidos/agonistas , Relaxina/análogos & derivados , Relaxina/farmacología , Secuencia de Aminoácidos , Animales , Enfermedades Cardiovasculares , Línea Celular Tumoral , Células HEK293 , Semivida , Humanos , Lipopéptidos/genética , Lipopéptidos/farmacocinética , Masculino , Simulación de Dinámica Molecular , Estructura Molecular , Mutación , Subunidades de Proteína , Ratas Sprague-Dawley , Relaxina/genética , Relación Estructura-ActividadRESUMEN
The F1 part of the chloroplast H+ adenosine triphosphate (ATP)-synthase (CF1) strongly interacts with tentoxin, a natural fungous cyclic tetrapeptide known to inhibit the chloroplast enzyme and not the mammalian mitochondrial enzyme. Whereas the synthesis or the hydrolysis of ATP requires the stepwise rotation of the protein rotor gamma within the (alphabeta)3 crown, only one molecule of tentoxin is needed to fully inhibit the complex. With the help of an original homology modeling technique, based on robust distance geometry protocols, we built a tridimensional model of the alpha3beta3gamma CF1) subcomplex (3200 esidues), in which we introduced three different nucleotide occupancies to check their possible influence on the tentoxin binding site. Simultaneous comparison of three available high-resolution X-ray structures of F1, performed with a local structural alignment search tool, led to characterizing common structural blocks and the distorsions experienced by the complex during the catalytic turnover. The common structural blocks were used as a starting point of the spinach CF1 structure rebuilding. Finally, tentoxin was docked into its putative binding site of the reconstructed structure. The docking method was initially validated in the mitochondrial enzyme by its ability to relocate nucleotides into their original position in the crystal. Tentoxin binding was found possible to the two alpha/beta interfaces associated with the empty and adenosine diphosphate (ADP)-loaded catalytic sites, but not to the one associated with the ATP-loaded site. These results suggest a mechanism of CF1 inhibition by one molecule of tentoxin, by the impossibility of the alpha/beta interface bearing tentoxin to pass through the ATP-loaded state.
Asunto(s)
ATPasas de Translocación de Protón de Cloroplastos/química , Inhibidores Enzimáticos/química , Modelos Moleculares , Péptidos Cíclicos/química , Secuencia de Aminoácidos , Animales , Ácido Aspártico/química , ATPasas de Translocación de Protón Bacterianas/química , Sitios de Unión , Bovinos , ATPasas de Translocación de Protón de Cloroplastos/metabolismo , Cristalografía por Rayos X , Inhibidores Enzimáticos/metabolismo , Imagenología Tridimensional , Sustancias Macromoleculares , ATPasas de Translocación de Protón Mitocondriales/química , Datos de Secuencia Molecular , Movimiento (Física) , Nucleótidos/metabolismo , Péptidos Cíclicos/metabolismo , Subunidades de Proteína , Ratas , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Spinacia oleracea/enzimología , Levaduras/enzimologíaRESUMEN
The mechanism of inhibition and reactivation of chloroplast ATP-synthase by the fungal cyclotetrapeptide tentoxin was investigated by photolabeling experiments, binding studies, and kinetic analysis using synthetic analogues of tentoxin. The alpha-subunit of chloroplast F(1)-ATPase (CF(1)) was specifically labeled by a photoactivatable tentoxin derivative, providing the first direct evidence of tentoxin binding to the alpha-subunit, and 3D homology modeling was used to locate tentoxin in its putative binding site at the alpha/beta interface. The non-photosynthetic F(1)-ATPase from thermophilic bacterium (TF(1)) proved to be also tentoxin-sensitive, and enzyme turnover dramatically increased the rate of tentoxin binding to its inhibitory site, contrary to what was previously observed with epsilon-depleted CF(1) [Santolini, J., Haraux, F., Sigalat, C., Moal, G., and André, F. (1999) J. Biol. Chem. 274, 849-858]. We propose that tentoxin preferentially binds to an ADP-loaded alpha beta pair, and mechanically blocks the catalytic cycle, perhaps by the impossibility of converting this alpha beta pair into an ATP-loaded alpha beta pair. Using (14)C-tentoxin and selected synthetic analogues, we found that toxin binding to the tight inhibitory site of CF(1) exerts some cooperative effect on the loose reactivatory site, but that no reciprocal effect exists. When the two tentoxin-binding sites are filled in reactivated F(1)-ATPase, they do not exchange their role during catalytic turnover, indicating an impairment between nucleotide occupancy and the shape of tentoxin-binding pocket. This analysis provides a mechanical interpretation of the inhibition of F(1)-ATPase by tentoxin and a clue for understanding the reactivation process.