RESUMEN
Photoactivated adenylate cyclases (PACs) are light activated enzymes that combine blue light sensing capacity with the ability to convert ATP to cAMP and pyrophosphate (PPi) in a light-dependent manner. In most of the known PACs blue light regulation is provided by a blue light sensing domain using flavin which undergoes a structural reorganization after blue-light absorption. This minor structural change then is translated toward the C-terminal of the protein, inducing a larger conformational change that results in the ATP conversion to cAMP. As cAMP is a key second messenger in numerous signal transduction pathways regulating various cellular functions, PACs are of great interest in optogenetic studies. The optimal optogenetic device must be "silent" in the dark and highly responsive upon light illumination. PAC from Oscillatoria acuminata is a very good candidate as its basal activity is very small in the dark and the conversion rates increase 20-fold upon light illumination. We studied the effect of replacing D67 to N, in the blue light using flavin domain. This mutation was found to accelerate the primary electron transfer process in the photosensing domain of the protein, as has been predicted. Furthermore, it resulted in a longer lived signaling state, which was formed with a lower quantum yield. Our studies show that the overall effects of the D67N mutation lead to a slightly higher conversion of ATP to cAMP, which points in the direction that by fine tuning the kinetic properties more responsive PACs and optogenetic devices can be generated.
Asunto(s)
Adenilil Ciclasas , Proteínas Bacterianas , Oscillatoria , Adenosina Trifosfato , Adenilil Ciclasas/genética , Adenilil Ciclasas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Flavinas/metabolismo , Luz , Sistemas de Mensajero Secundario , Oscillatoria/enzimologíaRESUMEN
InhA, the Mycobacterium tuberculosis enoyl-ACP reductase, is a target for the tuberculosis (TB) drug isoniazid (INH). InhA inhibitors that do not require KatG activation avoid the most common mechanism of INH resistance, and there are continuing efforts to fully elucidate the enzyme mechanism to drive inhibitor discovery. InhA is a member of the short-chain dehydrogenase/reductase superfamily characterized by a conserved active site Tyr, Y158 in InhA. To explore the role of Y158 in the InhA mechanism, this residue has been replaced by fluoroTyr residues that increase the acidity of Y158 up to â¼3200-fold. Replacement of Y158 with 3-fluoroTyr (3-FY) and 3,5-difluoroTyr (3,5-F2Y) has no effect on kcatapp/KMapp nor on the binding of inhibitors to the open form of the enzyme (Kiapp), whereas both kcatapp/KMapp and Kiapp are altered by seven-fold for the 2,3,5-trifluoroTyr variant (2,3,5-F3Y158 InhA). 19F NMR spectroscopy suggests that 2,3,5-F3Y158 is ionized at neutral pH indicating that neither the acidity nor ionization state of residue 158 has a major impact on catalysis or on the binding of substrate-like inhibitors. In contrast, Ki*app is decreased 6- and 35-fold for the binding of the slow-onset inhibitor PT504 to 3,5-F2Y158 and 2,3,5-F3Y158 InhA, respectively, indicating that Y158 stabilizes the closed form of the enzyme adopted by EI*. The residence time of PT504 is reduced â¼four-fold for 2,3,5-F3Y158 InhA compared to wild-type, and thus, the hydrogen bonding interaction of the inhibitor with Y158 is an important factor in the design of InhA inhibitors with increased residence times on the enzyme.
Asunto(s)
Mycobacterium tuberculosis , Tuberculosis , Humanos , Antituberculosos/farmacología , Antituberculosos/química , Isoniazida/química , Isoniazida/farmacología , Dominio Catalítico , Proteínas Bacterianas/químicaRESUMEN
The design of PROteolysis-TArgeting Chimeras (PROTACs) requires bringing an E3 ligase into proximity with a target protein to modulate the concentration of the latter through its ubiquitination and degradation. Here, we present a method for generating high-accuracy structural models of E3 ligase-PROTAC-target protein ternary complexes. The method is dependent on two computational innovations: adding a "silent" convolution term to an efficient protein-protein docking program to eliminate protein poses that do not have acceptable linker conformations and clustering models of multiple PROTACs that use the same E3 ligase and target the same protein. Results show that the largest consensus clusters always have high predictive accuracy and that the ensemble of models can be used to predict the dissociation rate and cooperativity of the ternary complex that relate to the degrading activity of the PROTAC. The method is demonstrated by applications to known PROTAC structures and a blind test involving PROTACs against BRAF mutant V600E. The results confirm that PROTACs function by stabilizing a favorable interaction between the E3 ligase and the target protein but do not necessarily exploit the most energetically favorable geometry for interaction between the proteins.
Asunto(s)
Proteínas , Ubiquitina-Proteína Ligasas , Proteolisis , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas/metabolismo , UbiquitinaciónRESUMEN
Light activated proteins are at the heart of photobiology and optogenetics, so there is wide interest in understanding the mechanisms coupling optical excitation to protein function. In addition, such light activated proteins provide unique insights into the real-time dynamics of protein function. Using pump-probe spectroscopy, the function of a photoactive protein can be initiated by a sub-100 fs pulse of light, allowing subsequent protein dynamics to be probed from femtoseconds to milliseconds and beyond. Among the most interesting photoactive proteins are the blue light using flavin (BLUF) domain proteins, which regulate the response to light of a wide range of bacterial and some euglenoid processes. The photosensing mechanism of BLUF domains has long been a subject of debate. In contrast to other photoactive proteins, the electronic and nuclear structure of the chromophore (flavin) is the same in dark- and light-adapted states. Thus, the driving force for photoactivity is unclear.To address this question requires real-time observation of both chromophore excited state processes and their effect on the structure and dynamics of the surrounding protein matrix. In this Account we describe how time-resolved infrared (IR) experiments, coupled with chemical biology, provide important new insights into the signaling mechanism of BLUF domains. IR measurements are sensitive to changes in both chromophore electronic structure and protein hydrogen bonding interactions. These contributions are resolved by isotope labeling of the chromophore and protein separately. Further, a degree of control over BLUF photochemistry is achieved through mutagenesis, while unnatural amino acid substitution allows us to both fine-tune the photochemistry and time resolve protein dynamics with spatial resolution.Ultrafast studies of BLUF domains reveal non-single-exponential relaxation of the flavin excited state. That relaxation leads within one nanosecond to the original flavin ground state bound in a modified hydrogen-bonding network, as seen in transient and steady-state IR spectroscopy. The change in H-bond configuration arises from formation of an unusual enol (imine) form of a critical glutamine residue. The dynamics observed, complemented by quantum mechanical calculations, suggest a unique sequential electron then double proton transfer reaction as the driving force, followed by rapid reorganization in the binding site and charge recombination. Importantly, studies of several BLUF domains reveal an unexpected diversity in their dynamics, although the underlying structure appears highly conserved. It is suggested that this diversity reflects structural dynamics in the ground state at standard temperature, leading to a distribution of structures and photochemical outcomes. Time resolved IR measurements were extended to the millisecond regime for one BLUF domain, revealing signaling state formation on the microsecond time scale. The mechanism involves reorganization of a ß-sheet connected to the chromophore binding pocket via a tryptophan residue. The potential of site-specific labeling amino acids with IR labels as a tool for probing protein structural dynamics was demonstrated.In summary, time-resolved IR studies of BLUF domains (along with related studies at visible wavelengths and quantum and molecular dynamics calculations) have resolved the photoactivation mechanism and real-time dynamics of signaling state formation. These measurements provide new insights into protein structural dynamics and will be important in optimizing the potential of BLUF domains in optobiology.
Asunto(s)
Proteínas Bacterianas , Flavinas , Proteínas Bacterianas/química , Transporte de Electrón , Flavinas/química , Enlace de Hidrógeno , Estructura Terciaria de ProteínaRESUMEN
Ceramides impact a diverse array of biological functions and have been implicated in disease pathogenesis. The enzyme neutral ceramidase (nCDase) is a zinc-containing hydrolase and mediates the metabolism of ceramide to sphingosine (Sph), both in cells and in the intestinal lumen. nCDase inhibitors based on substrate mimetics, for example C6-urea ceramide, have limited potency, aqueous solubility, and micelle-free fraction. To identify non-ceramide mimetic nCDase inhibitors, hit compounds from an HTS campaign were evaluated in biochemical, cell based and in silico modeling approaches. A majority of small molecule nCDase inhibitors contained pharmacophores capable of zinc interaction but retained specificity for nCDase over zinc-containing acid and alkaline ceramidases, as well as matrix metalloprotease-3 and histone deacetylase-1. nCDase inhibitors were refined by SAR, were shown to be substrate competitive and were active in cellular assays. nCDase inhibitor compounds were modeled by in silico DOCK screening and by molecular simulation. Modeling data supports zinc interaction and a similar compound binding pose with ceramide. nCDase inhibitors were identified with notably improved activity and solubility in comparison with the reference lipid-mimetic C6-urea ceramide.
Asunto(s)
Ceramidas , Ceramidasa Neutra , Dominio Catalítico , Ceramidas/química , Ceramidasa Neutra/antagonistas & inhibidores , Esfingosina/químicaRESUMEN
Tryptophan and tyrosine radical intermediates play crucial roles in many biological charge transfer processes. Particularly in flavoprotein photochemistry, short-lived reaction intermediates can be studied by the complementary techniques of ultrafast visible and infrared spectroscopy. The spectral properties of tryptophan radical are well established, and the formation of neutral tyrosine radicals has been observed in many biological processes. However, only recently, the formation of a cation tyrosine radical was observed by transient visible spectroscopy in a few systems. Here, we assigned the infrared vibrational markers of the cationic and neutral tyrosine radical at 1483 and 1502 cm-1 (in deuterated buffer), respectively, in a variant of the bacterial methyl transferase TrmFO, and in the native glucose oxidase. In addition, we studied a mutant of AppABLUF blue-light sensor domain from Rhodobacter sphaeroides in which only a direct formation of the neutral radical was observed. Our studies highlight the exquisite sensitivity of transient infrared spectroscopy to low concentrations of specific radicals.
Asunto(s)
Flavoproteínas/química , Radicales Libres/química , Espectrofotometría Infrarroja , Tirosina/química , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cationes/química , Flavoproteínas/metabolismo , Glucosa Oxidasa/química , Glucosa Oxidasa/metabolismo , Metiltransferasas/química , Metiltransferasas/genética , Metiltransferasas/metabolismo , Mutagénesis Sitio-Dirigida , Proteínas del Complejo del Centro de Reacción Fotosintética/química , Proteínas del Complejo del Centro de Reacción Fotosintética/genética , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Rhodobacter sphaeroides/metabolismoRESUMEN
Blue light absorbing flavoproteins play important roles in a variety of photobiological processes. Consequently, there have been numerous investigations of their excited state structure and dynamics, in particular by time-resolved vibrational spectroscopy. The isoalloxazine chromophore of the flavoprotein cofactors has been studied in detail by time-resolved Raman, lending it a benchmark status for mode assignments in excited electronic states of large molecules. However, detailed comparisons of calculated and measured spectra have proven challenging, as there are many more modes calculated than are observed, and the role of resonance enhancement is difficult to characterize in excited electronic states. Here we employ a recently developed approach due to Elles and co-workers ( J. Phys. Chem. A 2018, 122, 8308-8319) for the calculation of resonance-enhanced Raman spectra of excited states and apply it to the lowest singlet and triplet excited states of the isoalloxazine chromophore. There is generally good agreement between calculated and observed enhancements, which allows assignment of vibrational bands of the flavoprotein cofactors to be refined. However, some prominently enhanced bands are found to be absent from the calculations, suggesting the need for further development of the theory.
RESUMEN
Pluripotency is defined by the ability of a cell to differentiate to the derivatives of all the three embryonic germ layers: ectoderm, mesoderm and endoderm. Pluripotent cells can be captured via the archetypal derivation of embryonic stem cells or via somatic cell reprogramming. Somatic cells are induced to acquire a pluripotent stem cell (iPSC) state through the forced expression of key transcription factors, and in the mouse these cells can fulfil the strictest of all developmental assays for pluripotent cells by generating completely iPSC-derived embryos and mice. However, it is not known whether there are additional classes of pluripotent cells, or what the spectrum of reprogrammed phenotypes encompasses. Here we explore alternative outcomes of somatic reprogramming by fully characterizing reprogrammed cells independent of preconceived definitions of iPSC states. We demonstrate that by maintaining elevated reprogramming factor expression levels, mouse embryonic fibroblasts go through unique epigenetic modifications to arrive at a stable, Nanog-positive, alternative pluripotent state. In doing so, we prove that the pluripotent spectrum can encompass multiple, unique cell states.
Asunto(s)
Reprogramación Celular/genética , Reprogramación Celular/fisiología , Epigénesis Genética , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Animales , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Femenino , Fibroblastos/clasificación , Fibroblastos/citología , Fibroblastos/metabolismo , Histona Desacetilasas/metabolismo , Células Madre Pluripotentes Inducidas/clasificación , Ratones , Ratones Desnudos , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transgenes/genéticaRESUMEN
Somatic cell reprogramming to a pluripotent state continues to challenge many of our assumptions about cellular specification, and despite major efforts, we lack a complete molecular characterization of the reprograming process. To address this gap in knowledge, we generated extensive transcriptomic, epigenomic and proteomic data sets describing the reprogramming routes leading from mouse embryonic fibroblasts to induced pluripotency. Through integrative analysis, we reveal that cells transition through distinct gene expression and epigenetic signatures and bifurcate towards reprogramming transgene-dependent and -independent stable pluripotent states. Early transcriptional events, driven by high levels of reprogramming transcription factor expression, are associated with widespread loss of histone H3 lysine 27 (H3K27me3) trimethylation, representing a general opening of the chromatin state. Maintenance of high transgene levels leads to re-acquisition of H3K27me3 and a stable pluripotent state that is alternative to the embryonic stem cell (ESC)-like fate. Lowering transgene levels at an intermediate phase, however, guides the process to the acquisition of ESC-like chromatin and DNA methylation signature. Our data provide a comprehensive molecular description of the reprogramming routes and is accessible through the Project Grandiose portal at http://www.stemformatics.org.
Asunto(s)
Reprogramación Celular/genética , Genoma/genética , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Animales , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina , Metilación de ADN , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Epistasis Genética/genética , Fibroblastos/citología , Fibroblastos/metabolismo , Histonas/química , Histonas/metabolismo , Internet , Ratones , Proteoma/genética , Proteómica , ARN Largo no Codificante/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética/genética , Transcriptoma/genética , Transgenes/genéticaRESUMEN
N-Acyl sulfamoyladenosines (acyl-AMS) have been used extensively to inhibit adenylate-forming enzymes that are involved in a wide range of biological processes. These acyl-AMS inhibitors are nonhydrolyzable mimics of the cognate acyl adenylate intermediates that are bound tightly by adenylate-forming enzymes. However, the anionic acyl sulfamate moiety presents a pharmacological liability that may be detrimental to cell permeability and pharmacokinetic profiles. We have previously developed the acyl sulfamate OSB-AMS (1) as a potent inhibitor of the adenylate-forming enzyme MenE, an o-succinylbenzoate-CoA (OSB-CoA) synthetase that is required for bacterial menaquinone biosynthesis. Herein, we report the use of computational docking to develop novel, non-acyl sulfamate inhibitors of MenE. A m-phenyl ether-linked analogue (5) was found to be the most potent inhibitor (IC50 = 8 µM; Kd = 244 nM), and its X-ray co-crystal structure was determined to characterize its binding mode in comparison to the computational prediction. This work provides a framework for the development of potent non-acyl sulfamate inhibitors of other adenylate-forming enzymes in the future.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Proteínas de Escherichia coli/antagonistas & inhibidores , Succinato-CoA Ligasas/antagonistas & inhibidores , Vitamina K 2/metabolismo , Cristalografía por Rayos X , Diseño de Fármacos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Escherichia coli/enzimología , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Cinética , Pruebas de Sensibilidad Microbiana , Modelos Químicos , Simulación del Acoplamiento Molecular , Estructura Molecular , Mutación , Conformación Proteica , Succinato-CoA Ligasas/química , Succinato-CoA Ligasas/metabolismo , Ácidos Sulfónicos/química , Ácidos Sulfónicos/farmacologíaRESUMEN
The light, oxygen, voltage (LOV) domain proteins are blue light photoreceptors that utilize a noncovalently bound flavin mononucleotide (FMN) cofactor as the chromophore. The modular nature of these proteins has led to their wide adoption in the emerging fields of optogenetics and optobiology, where the LOV domain has been fused to a variety of output domains leading to novel light-controlled applications. In this work, we extend our studies of the subpicosecond to several hundred microsecond transient infrared spectroscopy of the isolated LOV domain AsLOV2 to three full-length photoreceptors in which the LOV domain is fused to an output domain: the LOV-STAS protein, YtvA, the LOV-HTH transcription factor, EL222, and the LOV-histidine kinase, LovK. Despite differences in tertiary structure, the overall pathway leading to cysteine adduct formation from the FMN triplet state is highly conserved, although there are slight variations in rate. However, significant differences are observed in the vibrational spectra and kinetics after adduct formation, which are directly linked to the specific output function of the LOV domain. While the rate of adduct formation varies by only 3.6-fold among the proteins, the subsequent large-scale structural changes in the full-length LOV photoreceptors occur over the micro- to submillisecond time scales and vary by orders of magnitude depending on the different output function of each LOV domain.
Asunto(s)
Fotorreceptores Microbianos/efectos de la radiación , Fotorreceptores de Plantas/efectos de la radiación , Espectroscopía Infrarroja por Transformada de Fourier/métodos , Sitios de Unión , Cristalografía por Rayos X , Cisteína/química , Mononucleótido de Flavina/química , Enlace de Hidrógeno , Modelos Moleculares , Fotoblanqueo , Fotoquímica , Fotorreceptores Microbianos/química , Fotorreceptores de Plantas/química , Conformación Proteica , Dominios Proteicos , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/efectos de la radiación , Técnica de SustracciónRESUMEN
Heterogeneity in pluripotent stem cell (PSC) aggregation leads to variability in mass transfer and signaling gradients between aggregates, which results in heterogeneous differentiation and therefore variability in product quality and yield. We have characterized a chemical-based method to control aggregate size within a specific, tunable range with low heterogeneity, thereby reducing process variability in PSC expansion. This method enables controlled, scalable, stirred suspension-based manufacturing of PSC cultures that are critical for the translation of regenerative medicine strategies to clinical products.
Asunto(s)
Biotecnología/métodos , Agregación Celular , Técnicas Citológicas/métodos , Células Madre Pluripotentes/fisiologíaRESUMEN
There is growing awareness of the link between drug-target residence time and in vivo drug activity, and there are increasing efforts to determine the molecular factors that control the lifetime of a drug-target complex. Rational alterations in the drug-target residence time require knowledge of both the ground and transition states on the inhibition reaction coordinate, and we have determined the structure-kinetic relationship for 22 ethyl- or hexyl-substituted diphenyl ethers that are slow-binding inhibitors of bpFabI1, the enoyl-ACP reductase FabI1 from Burkholderia pseudomallei. Analysis of enzyme inhibition using a two-dimensional kinetic map demonstrates that the ethyl and hexyl diphenyl ethers fall into two distinct clusters. Modifications to the ethyl diphenyl ether B ring result in changes to both on and off rates, where residence times of up to â¼700 min (â¼11 h) are achieved by either ground state stabilization (PT444) or transition state destabilization (slower on rate) (PT404). By contrast, modifications to the hexyl diphenyl ether B ring result in residence times of 300 min (â¼5 h) through changes in only ground state stabilization (PT119). Structural analysis of nine enzyme:inhibitor complexes reveals that the variation in structure-kinetic relationships can be rationalized by structural rearrangements of bpFabI1 and subtle changes to the orientation of the inhibitor in the binding pocket. Finally, we demonstrate that three compounds with residence times on bpFabI1 from 118 min (â¼2 h) to 670 min (â¼11 h) have in vivo efficacy in an acute B. pseudomallei murine infection model using the virulent B. pseudomallei strain Bp400.
Asunto(s)
Antibacterianos/química , Proteínas Bacterianas/antagonistas & inhibidores , Burkholderia pseudomallei/efectos de los fármacos , Enoil-ACP Reductasa (NADH)/antagonistas & inhibidores , Inhibidores Enzimáticos/química , Melioidosis/dietoterapia , Éteres Fenílicos/química , Animales , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Burkholderia pseudomallei/enzimología , Burkholderia pseudomallei/genética , Burkholderia pseudomallei/crecimiento & desarrollo , Recuento de Colonia Microbiana , Cristalografía por Rayos X , Enoil-ACP Reductasa (NADH)/genética , Enoil-ACP Reductasa (NADH)/metabolismo , Inhibidores Enzimáticos/farmacología , Femenino , Expresión Génica , Cinética , Pulmón/efectos de los fármacos , Pulmón/microbiología , Melioidosis/tratamiento farmacológico , Melioidosis/microbiología , Ratones , Ratones Endogámicos BALB C , Pruebas de Sensibilidad Microbiana , Simulación de Dinámica Molecular , Éteres Fenílicos/farmacología , Unión Proteica , Estructura Secundaria de Proteína , Bazo/efectos de los fármacos , Bazo/microbiología , Relación Estructura-ActividadRESUMEN
The flavin chromophore in blue-light-using FAD (BLUF) photoreceptors is surrounded by a hydrogen bond network that senses and responds to changes in the electronic structure of the flavin on the ultrafast time scale. The hydrogen bond network includes a strictly conserved Tyr residue, and previously we explored the role of this residue, Y21, in the photoactivation mechanism of the BLUF protein AppABLUF by the introduction of fluorotyrosine (F-Tyr) analogues that modulated the pKa and reduction potential of Y21 by 3.5 pH units and 200 mV, respectively. Although little impact on the forward (dark- to light-adapted form) photoreaction was observed, the change in Y21 pKa led to a 4000-fold increase in the rate of dark-state recovery. In the present work we have extended these studies to the BLUF protein PixD, where, in contrast to AppABLUF, modulation in the Tyr (Y8) pKa has a profound impact on the forward photoreaction. In particular, a decrease in Y8 pKa by 2 or more pH units prevents formation of a stable light state, consistent with a photoactivation mechanism that involves proton transfer or proton-coupled electron transfer from Y8 to the electronically excited FAD. Conversely, the effect of pKa on the rate of dark recovery is markedly reduced in PixD. These observations highlight very significant differences between the photocycles of PixD and AppABLUF, despite their sharing highly conserved FAD binding architectures.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/efectos de la radiación , Flavoproteínas/metabolismo , Flavoproteínas/efectos de la radiación , Flúor/metabolismo , Luz , Fotorreceptores Microbianos/metabolismo , Fotorreceptores Microbianos/efectos de la radiación , Tirosina/metabolismo , Sitios de Unión , Color , Transporte de Electrón , Flavina-Adenina Dinucleótido/metabolismo , Enlace de Hidrógeno , Concentración de Iones de Hidrógeno , Dominios Proteicos , Protones , Synechocystis/químicaRESUMEN
A critical goal of lead compound selection and optimization is to maximize target engagement while minimizing off-target binding. Since target engagement is a function of both the thermodynamics and kinetics of drug-target interactions, it follows that the structures of both the ground states and transition states on the binding reaction coordinate are needed to rationally modulate the lifetime of the drug-target complex. Previously, we predicted the structure of the rate-limiting transition state that controlled the time-dependent inhibition of the enoyl-ACP reductase InhA. This led to the discovery of a triazole-containing diphenyl ether with an increased residence time on InhA due to transition-state destabilization rather than ground-state stabilization. In the present work, we evaluate the inhibition of InhA by 14 triazole-based diphenyl ethers and use a combination of enzyme kinetics and X-ray crystallography to generate a structure-kinetic relationship for time-dependent binding. We show that the triazole motif slows the rate of formation for the final drug-target complex by up to 3 orders of magnitude. In addition, we identify a novel inhibitor with a residence time on InhA of 220 min, which is 3.5-fold longer than that of the INH-NAD adduct formed by the tuberculosis drug, isoniazid. This study provides a clear example in which the lifetime of the drug-target complex is controlled by interactions in the transition state for inhibitor binding rather than the ground state of the enzyme-inhibitor complex, and demonstrates the important role that on-rates can play in drug-target residence time.
Asunto(s)
Inhibinas/antagonistas & inhibidores , Termodinámica , Triazoles/farmacología , Cristalografía por Rayos X , Humanos , Inhibinas/metabolismo , Cinética , Modelos Moleculares , Estructura Molecular , Factores de Tiempo , Triazoles/químicaRESUMEN
Many drug candidates fail in clinical trials owing to a lack of efficacy from limited target engagement or an insufficient therapeutic index. Minimizing off-target effects while retaining the desired pharmacodynamic (PD) response can be achieved by reduced exposure for drugs that display kinetic selectivity in which the drug-target complex has a longer half-life than off-target-drug complexes. However, though slow-binding inhibition kinetics are a key feature of many marketed drugs, prospective tools that integrate drug-target residence time into predictions of drug efficacy are lacking, hindering the integration of drug-target kinetics into the drug discovery cascade. Here we describe a mechanistic PD model that includes drug-target kinetic parameters, including the on- and off-rates for the formation and breakdown of the drug-target complex. We demonstrate the utility of this model by using it to predict dose response curves for inhibitors of the LpxC enzyme from Pseudomonas aeruginosa in an animal model of infection.
Asunto(s)
Amidohidrolasas/antagonistas & inhibidores , Antibacterianos/farmacología , Inhibidores Enzimáticos/farmacología , Ácidos Hidroxámicos/farmacología , Treonina/análogos & derivados , Animales , Antibacterianos/química , Antibacterianos/farmacocinética , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacocinética , Ácidos Hidroxámicos/química , Ácidos Hidroxámicos/farmacocinética , Cinética , Ratones Endogámicos , Pruebas de Sensibilidad Microbiana , Modelos Biológicos , Estructura Molecular , Unión Proteica , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/enzimología , Treonina/química , Treonina/farmacocinética , Treonina/farmacología , Factores de TiempoRESUMEN
The enoyl-ACP reductase (ENR) catalyzes the last reaction in the elongation cycle of the bacterial type II fatty acid biosynthesis (FAS-II) pathway. While the FabI ENR is a well-validated drug target in organisms such as Mycobacterium tuberculosis and Staphylococcus aureus, alternate ENR isoforms have been discovered in other pathogens, including the FabV enzyme that is the sole ENR in Yersinia pestis (ypFabV). Previously, we showed that the prototypical ENR inhibitor triclosan was a poor inhibitor of ypFabV and that inhibitors based on the 2-pyridone scaffold were more potent [Hirschbeck, M. (2012) Structure 20 (1), 89-100]. These studies were performed with the T276S FabV variant. In the work presented here, we describe a detailed examination of the mechanism and inhibition of wild-type ypFabV and the T276S variant. The T276S mutation significantly reduces the affinity of diphenyl ether inhibitors for ypFabV (20-fold â 100-fold). In addition, while T276S ypFabV generally displays an affinity for 2-pyridone inhibitors higher than that of the wild-type enzyme, the 4-pyridone scaffold yields compounds with similar affinity for both wild-type and T276S ypFabV. T276 is located at the N-terminus of the helical substrate-binding loop, and structural studies coupled with site-directed mutagenesis reveal that alterations in this residue modulate the size of the active site portal. Subsequently, we were able to probe the mechanism of time-dependent inhibition in this enzyme family by extending the inhibition studies to include P142W ypFabV, a mutation that results in a gain of slow-onset inhibition for the 4-pyridone PT156.
Asunto(s)
Enoil-ACP Reductasa (NADH)/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Éteres Fenílicos/química , Piridonas/química , Yersinia pestis/enzimología , Catálisis , Dominio Catalítico , Cristalización , Cristalografía por Rayos X , Enoil-ACP Reductasa (NADH)/genética , Enoil-ACP Reductasa (NADH)/metabolismo , Modelos Moleculares , Simulación de Dinámica Molecular , Mutagénesis Sitio-Dirigida , Mutación/genética , NAD/metabolismo , Unión Proteica , Conformación ProteicaRESUMEN
The transcriptional antirepressor AppA is a blue light using flavin (BLUF) photoreceptor that releases the transcriptional repressor PpsR upon photoexcitation. Light activation of AppA involves changes in a hydrogen-bonding network that surrounds the flavin chromophore on the nanosecond time scale, while the dark state of AppA is then recovered in a light-independent reaction with a dramatically longer half-life of 15 min. Residue Y21, a component of the hydrogen-bonding network, is known to be essential for photoactivity. Here, we directly explore the effect of the Y21 pKa on dark state recovery by replacing Y21 with fluorotyrosine analogues that increase the acidity of Y21 by 3.5 pH units. Ultrafast transient infrared measurements confirm that the structure of AppA is unperturbed by fluorotyrosine substitution, and that there is a small (3-fold) change in the photokinetics of the forward reaction over the fluorotyrosine series. However, reduction of 3.5 pH units in the pKa of Y21 increases the rate of dark state recovery by 4000-fold with a Brønsted coefficient of â¼ 1, indicating that the Y21 proton is completely transferred in the transition state leading from light to dark adapted AppA. A large solvent isotope effect of â¼ 6-8 is also observed on the rate of dark state recovery. These data establish that the acidity of Y21 is a crucial factor for stabilizing the light activated form of the protein, and have been used to propose a model for dark state recovery that will ultimately prove useful for tuning the properties of BLUF photosensors for optogenetic applications.