15 research outputs found
Probing the Kinetic and Thermodynamic Fingerprints of Anti-EGF Nanobodies by Surface Plasmon Resonance
Despite the widespread use of antibodies in clinical applications, the precise molecular mechanisms underlying antibody-antigen (Ab-Ag) interactions are often poorly understood. In this study, we exploit the technical features of a typical surface plasmon resonance (SPR) biosensor to dissect the kinetic and thermodynamic components that govern the binding of single-domain Ab or nanobodies to their target antigen, epidermal growth factor (EGF), a key oncogenic protein that is involved in tumour progression. By carefully tuning the experimental conditions and transforming the kinetic data into equilibrium constants, we reveal the complete picture of binding thermodynamics, including the energetics of the complex-formation transition state. This approach, performed using an experimentally simple and high-throughput setup, is expected to facilitate mechanistic studies of Ab-based therapies and, importantly, promote the rational development of new biological drugs with suitable properties
Toward a novel drug to target the EGF-EGFR interaction: design of metabolically stable bicyclic peptides
In cancer, proliferation of malignant cells is driven by overactivation of growth-signalling mechanisms, such as the epidermal growth factor receptor (EGFR) pathway. Despite its therapeutic relevance, the EGF-EGFR interaction has remained elusive to inhibition by synthetic molecules, mostly as a result of its large size and lack of binding pockets and cavities. Designed peptides, featuring cyclic motifs and other structural constraints, have the potential to modulate such challenging protein-protein interactions (PPIs). Herein, we present the structure-based design of a series of bicyclic constrained peptides that mimic an interface domain of EGFR and inhibit the EGF-EGFR interaction by targeting the smaller partner (i.e., EGF). This design process was guided by the integrated use of in silico methods and biophysical techniques, such as NMR spectroscopy and surface acoustic wave. The best analogues were able to reduce selectively the viability of EGFR+ human cancer cells. In addition to their efficacy, these bicyclic peptides are endowed with exceptional stability and metabolic resistance-two features that make them suitable candidates for in vivo applications
The Pseudomonas aeruginosa substrate-binding protein Ttg2D functions as a general glycerophospholipid transporter across the periplasm
In Pseudomonas aeruginosa, Ttg2D is the soluble periplasmic phospholipid-binding component of an ABC transport system thought to be involved in maintaining the asymmetry of the outer membrane. Here we use the crystallographic structure of Ttg2D at 2.5 Å resolution to reveal that this protein can accommodate four acyl chains. Analysis of the available structures of Ttg2D orthologs shows that they conform a new substrate-binding-protein structural cluster. Native and denaturing mass spectrometry experiments confirm that Ttg2D, produced both heterologously and homologously and isolated from the periplasm, can carry two diacyl glycerophospholipids as well as one cardiolipin. Binding is notably promiscuous, allowing the transport of various molecular species. In vitro binding assays coupled to native mass spectrometry show that binding of cardiolipin is spontaneous. Gene knockout experiments in P. aeruginosa multidrug-resistant strains reveal that the Ttg2 system is involved in low-level intrinsic resistance against certain antibiotics that use a lipid-mediated pathway to permeate through membranes
Specific expression pattern of tissue cytokines analyzed through the Surface Acoustic Wave technique is associated with age-related spontaneous beign prostatic hyperplasia in rats
The aim of the study reported herein was to evaluate the suitability of the Surface Acoustic Wave (SAW) technique as a possible diagnostic tool in benign prostatic hyperplasia (BPH). Moreover, for the first time, the BPH model was a totally physiological using naturally aged rats with spontaneous, age-related BPH instead of the pharmacologically induced models usually used. Eighteen male Wistar rats were distributed according to their age: 6 weeks (young), 12 weeks (adult) and 12 months (old) old. Prostate gland was removed and analyzed by mini-arrays, Western blotting (WB) and SAW techniques. Mini-arrays indicated that there were significant differences in the expression of 29/34 inflammation-related cytokines. WB was carried out to confirm the results after selection of 4 cytokines from which one showed no changes, namely PDGF-AA, and the other three, which significantly increase in older animals, were CD86, β-NGF and VEGF. Notwithstanding, WB of old rats yielded confusing results due to an anomalous migration of proteins, dismissing this technique as an useful tool in these animals. Accurate results in old rats were uniquely obtained by using the SAW technique. Thus, SAW analysis showed that there were not differences among groups in the amount of PDGF-AA. On the contrary, SAW analysis showed that amounts of CD86, β-NGF and VEGF in old rats were 2.0, 1.9 and 5.7-fold higher than that from young ones, respectively. These results indicate that SAW is a highly accurate technique for determining changes in the cytokines expression in BPH
N-lauroylation during the expression of recombinant N- myristoylated proteins: implications and solutions
Incorporation of myristic acid to the N-terminus of proteins is a crucial modification that promotes membrane binding and proper localization of important components of signaling pathways. Recombinant expression of N-myristoylatyed proteins in E. coli can be achieved by co-expressing yeast N-myristoyltransferase and supplementing the growth medium with myristic acid. However, undesired incorporation of the 12-carbon fatty acid lauric acid can occur (leading to heterogeneous samples), especially when the available carbon sources are scarce, as it is the case in minimal medium for the expression of isotopically enriched samples. By applying this method to the Brain-acid soluble protein 1 and the 1- 185 N-terminal region of c-Src, we show the significant, and protein- specific, differences in the membrane binding properties of lauroylated and myristoylated forms. We also present a robust strategy for obtaining lauryl free-samples of myristoylated proteins in both rich and minimal media
Effectiveness of Fosfomycin for the Treatment of Multidrug-Resistant Escherichia coli Bacteremic Urinary Tract Infections
IMPORTANCE The consumption of broad-spectrum drugs has increased as a consequence of the spread of multidrug-resistant (MDR) Escherichia coli. Finding alternatives for these infections is critical, for which some neglected drugs may be an option. OBJECTIVE To determine whether fosfomycin is noninferior to ceftriaxone or meropenem in the targeted treatment of bacteremic urinary tract infections (bUTIs) due to MDR E coli. DESIGN, SETTING, AND PARTICIPANTS This multicenter, randomized, pragmatic, open clinical trial was conducted at 22 Spanish hospitals from June 2014 to December 2018. Eligible participants were adult patients with bacteremic urinary tract infections due to MDR E coli; 161 of 1578 screened patients were randomized and followed up for 60 days. Data were analyzed in May 2021. INTERVENTIONS Patients were randomized 1 to 1 to receive intravenous fosfomycin disodium at 4 g every 6 hours (70 participants) or a comparator (ceftriaxone or meropenem if resistant; 73 participants) with the option to switch to oral fosfomycin trometamol for the fosfomycin group or an active oral drug or pa renteral ertapenem for the comparator group after 4 days. MAIN OUTCOMES AND MEASURES The primary outcome was clinical and microbiological cure (CMC) 5 to 7 days after finalization of treatment; a noninferiority margin of 7% was considered. RESULTS Among 143 patients in the modified intention-to-treat population (median [IQR] age, 72 [62-81] years; 73 [51.0%] women), 48 of 70 patients (68.6%) treated with fosfomycin and 57 of 73 patients (78.1%) treated with comparators reached CMC (risk difference, -9.4 percentage points; 1-sided 95% CI, -21.5 to infinity percentage points; P = .10). While clinical or microbiological failure occurred among 10 patients (14.3%) treated with fosfomycin and 14 patients (19.7%) treated with comparators (risk difference, -5.4 percentage points; 1-sided 95% CI. -infinity to 4.9; percentage points; P = .19), an increased rate of adverse event-related discontinuations occurred with fosfomycin vs comparators (6 discontinuations [8.5%] vs 0 discontinuations; P = .006). In an exploratory analysis among a subset of 38 patients who underwent rectal colonization studies, patients treated with fosfomycin acquired a new ceftriaxone-resistant or meropenem-resistant gram-negative bacteria at a decreased rate compared with patients treated with comparators (0 of 21 patients vs 4 of 17 patients [23.5%]; 1-sided P = .01). CONCLUSIONS AND RELEVANCE This study found that fosfomycin did not demonstrate noninferiority to comparators as targeted treatment of bUTI from MDR E coli; this was due to an increased rate of adverse event-related discontinuations. This finding suggests that fosfomycin may be considered for selected patients with these infections
Estudi del mecanisme catalític de les glicosiltransferases que operen amb retenció de configuració
[cat] Estudis previs basats en l’alineament de seqüències, la predicció d’estructures i la superposició d’estructures resoltes, han demostrat que les glicogen sintases (GSs) d’animals i fongs de la família 3 de les glicosiltransferases (GTs), i les GSs de bacteris i midó sintases de plantes, membres de la família 5 de les GTs, tenen en comú un conjunt de característiques estructurals que fan suposar que tots aquests enzims operen a través d’un mecanisme catalític molt similar. Mentre que el mecanisme de desplaçament directe proposat per a les GTs que operen amb inversió de configuració ha obtingut suport experimental i teòric, el mecanisme catalític de les GTs que operen amb retenció de configuració no està gens clar i, fins al moment, diferents autors han arribat a proposar diversos mecanismes diferents, però cap d’ells ha obtingut alhora suport experimental i teòric, quedant moltes qüestions pendents per resoldre. Per obtenir més informació referent al mecanisme catalític de les GTs que operen amb retenció de configuració, en aquesta tesi és van seguir dues estratègies. Primerament, a partir de la co-cristal•lització de l’enzim glicogen sintasa de Pyrococcus abyssi (PaGS) amb UDP-glucosa com a donador de glicosil, en absència de substrat acceptor, es va observar que l’enzim descomponia el substrat donador. En aquesta tesi es s’ha identificar el producte obtingut en el centre actiu que és una molècula de d’1,5-anhidro-D-arabino-hexa-1-enitol que tautomeritza cap a la forma més estable 1,5-anhidro-D-fructosa. Malauradament, no s’ha pogut demostrar la competència catalítica de la 1,5-anhidrofuctosa com a intermedi de la reacció de transferència glicosil catalitzada per PaGS (família GT-5) i la glicogenina de múscul de conill (GGN, família GT-8). Una de les estratègies utilitzades per poder trobar evidències sobre el mecanisme catalític dels enzims, es fer ús de compostos que puguin unir-se de manera no covalent als centres actius i a la vegada no siguin fàcilment degradables pels enzims. Per tal de poder obtenir estructures tridimensionals de complexes ternaris que ens permetin descriure la geometria del centre actiu en el procés catalític. Però actualment hi ha escassos inhibidors de glicosiltransferases que compleixin aquestes característiques. Per aquest motiu, en la segona estratègia d’aquesta tesi, es van estudiar compostos glicomimetics i pseudosucres com a plausibles inhibidors de GTs que operen amb retenció de configuració i, posteriorment, es van determinar els mecanismes cinètics d’inhibició. A més, estàvem especialment interessats en identificar inhibidors de dues glicosiltransferases: la glicogen sintasa i la glicogen fosforilasa de mamífer. S’ha descrit la glicogen sintasa com un enzim clau en certes malalties causades pel mal funcionament del metabolisme de glicogen i, en particular, en aquelles on es produeix una acumulació de glicogen aberrant en les neurones. D’altra banda, la glicogen fosforilasa també s’ha proposat com a enzim diana per combatre la diabetis mellitus no dependent d’insulina o tipus 2 (NIDDM). Per aquesta part de la tesi és va decidir treballar amb la glicogen fosforilasa de múscul de conill a (RMGPa, família GT-35) com a model de GT de tipus no-Leloir que utilitza glicogen com a substrat acceptor, la glicogen sintasa d’Escherichia coli (EcGS, família GT-5) com a model de GT tipus Leloir que també utilitza glicogen com a substrat acceptor i, finalment, la isoforma 4 de la sacarosa sintasa (SuSy 4) de Solanum Tuberosum L. (família GT-4) com un altre model de GT tipus Leloir que enlloc d’utilitzar un polímer com a substrat acceptor fa ús d’una molècula de fructosa, un monosacàrid, en la direcció de síntesi de sacarosa
Química al laboratori: una (r)evolució històrica
La química ens envolta. Mirem on mirem, sempre la trobem present: des que ens aixequem al matí i ens fem el cafè fins que ens n'anem a dormir i apaguem el llum de l'habitació. Però tota aquesta química que ens facilita la vida diària no seria possible si abans algú no l'hagués estudiat. La història de la química està marcada per uns
experiments concrets i uns personatges excepcionals: Van Helmont, Hales, Lavoisier, Volta, Fraunhofer i Bunsen, entre d'altres. Per això, convidem els lectors a endinsar-se en la fascinant història de la química i, amb l'ajut d'alguns experiments, recórrer els salts més importants de la química que han fet avançar la nostra societat. Al mateix temps, aquests experiments poden ser una eina didàctica per al professorat de secundària per introduir els alumnes
en el món de la química i en la història de la química, alhora que per despertar-los l'interès per aquesta ciència.Chemistry is around us. Everywhere we find it: when we wake up every morning and make our coffee until we go to sleep and turn off the light of our bedroom. However, all this chemistry that makes our daily life easier would not be possible if anyone had not studied it before. The history of chemistry is marked by specific experiments and
exceptional characters: Van Helmont, Hales, Lavoisier, Volta, Fraunhofer and Bunsen, among others. Therefore, we invite readers to explore the fascinating history of chemistry and, with the help of some experiments, travel through the most important jumps in chemistry that have made advance our society. At the same time, these
experiments can be a teaching tool for high school teachers to introduce students to the world of chemistry and the history of chemistry, and arouse their interest in this science
Probing the Kinetic and Thermodynamic Fingerprints of Anti-EGF Nanobodies by Surface Plasmon Resonance
Despite the widespread use of antibodies in clinical applications, the precise molecular mechanisms underlying antibody–antigen (Ab–Ag) interactions are often poorly understood. In this study, we exploit the technical features of a typical surface plasmon resonance (SPR) biosensor to dissect the kinetic and thermodynamic components that govern the binding of single-domain Ab or nanobodies to their target antigen, epidermal growth factor (EGF), a key oncogenic protein that is involved in tumour progression. By carefully tuning the experimental conditions and transforming the kinetic data into equilibrium constants, we reveal the complete picture of binding thermodynamics, including the energetics of the complex-formation transition state. This approach, performed using an experimentally simple and high-throughput setup, is expected to facilitate mechanistic studies of Ab-based therapies and, importantly, promote the rational development of new biological drugs with suitable properties
Selective photoregulation of the activity of glycogen synthase and glycogen phosphorylase, two key enzymes in glycogen metabolism
This journal is © The Royal Society of Chemistry 2015. Glycogen is a polymer of α-1,4- and α-1,6-linked glucose units that provides a readily available source of energy in living organisms. Glycogen synthase (GS) and glycogen phosphorylase (GP) are the two enzymes that control, respectively, the synthesis and degradation of this polysaccharide and constitute adequate pharmacological targets to modulate cellular glycogen levels, by means of inhibition of their catalytic activity. Here we report on the synthesis and biological evaluation of a selective inhibitor that consists of an azobenzene moiety glycosidically linked to the anomeric carbon of a glucose molecule. In the ground state, the more stable (E)-isomer of the azobenzene glucoside had a slight inhibitory effect on rat muscle GP (RMGP, IC50 = 4.9 mM) and Escherichia coli GS (EcGS, IC50 = 1.6 mM). After irradiation and subsequent conversion to the (Z)-form, the inhibitory potency of the azobenzene glucoside did not significantly change for RMGP (IC50 = 2.4 mM), while its effect on EcGS increased 50-fold (IC50 = 32 μM). Sucrose synthase 4 from potatoes, a glycosyltransferase that does not operate on glycogen, was only slightly inhibited by the (E)-isomer (IC50 = 0.73 mM). These findings could be rationalized on the basis of kinetic and computer-aided docking analysis, which indicated that both isomers of the azobenzene glucoside mimic the EcGS acceptor substrate and exert their inhibitory effect by binding to the glycogen subsite in the active center of the enzyme. The ability to selectively photoregulate the catalytic activity of key enzymes of glycogen metabolism may represent a new approach for the treatment of glycogen metabolism disorders.This work was supported by the Dirección General de Investigación, Ministerio de Ciencia y Tecnología, Spain, grants CTQ2006-00743 (to J. C. F.), BFU2011-30554 (to J. J. G.), and CTQ2012-36074 (to D. V.) and the CIBER de Diabetes y Enfermedades Metabólicas. M. Díaz-Lobo is grateful for the doctoral grant BES-2009-029354 from the Ministerio de Ciencia e Investigación. J. Garcia-Amorós is grateful for a Beatriu de Pinós postdoctoral grant from the Generalitat de Catalunya (Spain, 2011 BP-A-00270)Peer Reviewe