RESUMEN
Here we present a split-enzyme sensor approach for the sequence-specific detection of metal-based drug adducts of DNA. Split ß-lactamase reporters were constructed using domain A of the High Mobility Group Box 1 protein (HMGB1a) in conjunction with zinc finger DNA-binding domains. As a proof of concept, the sensors were characterized with the well-known drug cisplatin, which forms 1,2-intrastrand crosslinks with DNA that are recognized by HMGB1a. After promising results with cisplatin, five ruthenium-based drugs were studied, four of which produced significant signal over background. These results highlight the utility of our approach for rapid screening of novel metal-based chemotherapeutic drug candidates and provide evidence that HMGB1a likely binds to DNA adducts formed by NAMI-A (imidazolium trans-tetrachlorodimethylsulfoxideimidazoleruthenate(III)), KP1019 (indazolium trans-tetrachlorodiindazoleruthenate(III)), KP418 (imidazolium trans-tetrachlorodiimidazoleruthenate(III)), and RAPTA-C (dichloro(η6-p-cymene)(1,3,5-triaza-7-phosphaadamantane)ruthenium(II)). These results thus imply a potential biologically relevant mode of action for the ruthenium-based drugs investigated herein.
Asunto(s)
Antineoplásicos , Compuestos Organometálicos , Rutenio , Antineoplásicos/farmacología , Cisplatino/farmacología , ADN/química , Aductos de ADN , Compuestos Organometálicos/química , Rutenio/química , Compuestos de Rutenio , beta-LactamasasRESUMEN
We have recently developed a fragment based selection strategy for targeting kinases, where a small molecule warhead can be non-covalently tethered to a phage-displayed library of peptides. This approach was applied to the conversion of the promiscuous kinase inhibitor, staurosporine, into a potent bivalent ligand for cAMP-dependent protein kinase (PKA). Herein we report a systematic evaluation of this new bivalent ligand (BL); (a) Lineweaver-Burke analysis revealed that the BL, unlike substrate-based bivalent kinase inhibitors, displayed non-competitive inhibition with respect to the peptide substrate, suggesting an allosteric mechanism of action; (b) linker optimization of the BL, afforded one of the most potent, sub-nanomolar, inhibitors of PKA reported to date; (c) the BL was found to be modular, where attachment of active site targeted small molecule warheads in lieu of staurosporine could achieve similar gains in affinity; and (d) profiling studies of both the staurosporine derivative and the BL (amide isostere) against a panel of 90 kinases revealed almost unique enhancement in selectivity against PKA (>5-fold) compared to the starting staurosporine derivative. These combined results provide new insights for BL discovery, which has the potential to provide guidance toward the development of kinase selective reagents while uncovering new allosteric sites on kinases for therapeutic targeting.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/química , Inhibidores de Proteínas Quinasas/química , Estaurosporina/química , Sitio Alostérico , Dominio Catalítico , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Humanos , Cinética , Ligandos , Biblioteca de Péptidos , Inhibidores de Proteínas Quinasas/farmacología , Estaurosporina/farmacologíaRESUMEN
We have developed a multiweek laboratory project in which students isolate myoglobin and characterize its structure, function, and redox state. The important laboratory techniques covered in this project include size-exclusion chromatography, electrophoresis, spectrophotometric titration, and FTIR spectroscopy. Regarding protein structure, students work with computer modeling and visualization of myoglobin and its homologues, after which they spectroscopically characterize its thermal denaturation. Students also study protein function (ligand binding equilibrium) and are instructed on topics in data analysis (calibration curves, nonlinear vs. linear regression). This upper division biochemistry laboratory project is a challenging and rewarding one that not only exposes students to a wide variety of important biochemical laboratory techniques but also ties those techniques together to work with a single readily available and easily characterized protein, myoglobin.
Asunto(s)
Bioquímica/educación , Bioquímica/métodos , Modelos Moleculares , Mioglobina , Humanos , Mioglobina/química , Mioglobina/aislamiento & purificación , Mioglobina/metabolismo , Relación Estructura-ActividadRESUMEN
We employed the ArcGIS plug-in package PRO-GRADE (Lin et al. 2009), developed for zonation of recharge/discharge (R/D) for modeling two-dimensional aquifer systems, to develop alternative R/D zonations for an existing three-dimensional groundwater flow model of a complex hydrogeologic setting. Our process began by intersecting PRO-GRADE output with the existing model's 4-zone R/D representation to develop a model having 12 R/D zones (R12) and then calibrating the resulting model using PEST. We then revised the R12 zonation using supplementary GIS data to develop a 51-zone R/D zonation (R51). From R51, we developed a series of daughter models having 40, 30, 28, and 18 R/D zones by removing zones from R51 if calibration resulted in little change in the zone's starting R/D rate and/or if the model was insensitive to the zone's R/D rate. For these models (R40N, R30N, R28N, and R18N), we used the ArcGIS Nibble tool to rapidly and consistently reassign model cells within eliminated zones of R51 to the zone of the nearest model cell in a retained zone having the same starting value. R12, R51, R40N, R30N, R28N, and R18N are all more accurate than the original model (R4), although improvements relative to stream discharge targets exceeded improvements relative to head targets. The models also executed with better numerical stability and less mass balance discrepancy than R4. These improvements demonstrate that R/D estimation in a complex shallow three-dimensional steady-state model can be improved with PRO-GRADE estimates of R/D when guided by calibration statistics and supplemental geographic data.
Asunto(s)
Agua Subterránea , Modelos Teóricos , CalibraciónRESUMEN
The widespread success of affinity tags throughout the biological sciences has prompted interest in developing new and convenient labeling strategies. Affinity tags are well-established tools for recombinant protein immobilization and purification. More recently these tags have been utilized for selective biological targeting towards multiplexed protein detection in numerous imaging applications as well as for drug-delivery. Recently, we discovered a phage-display selected cyclic peptide motif that was shown to bind selectively to NeutrAvidin and avidin but not to the structurally similar streptavidin. Here, we have exploited this selectivity to develop an affinity tag based on the evolved DRATPY moiety that is orthogonal to known Strep-tag technologies. As proof of principle, the divalent AviD-tag (Avidin-Di-tag) was expressed as a Green Fluorescent Protein variant conjugate and exhibited superior immobilization and elution characteristics to the first generation Strep-tag and a monovalent DRATPY GFP-fusion protein analogue. Additionally, we demonstrate the potential for a peptide based orthogonal labeling strategy involving our divalent AviD-tag in concert with existing streptavidin-based affinity reagents. We believe the AviD-tag and its unique recognition properties will provide researchers with a useful new affinity reagent and tool for a variety of applications in the biological and chemical sciences.
Asunto(s)
Marcadores de Afinidad/química , Avidina/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Secuencia de Aminoácidos , Clonación Molecular , Epítopos/química , Escherichia coli/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Recombinantes de Fusión/químicaRESUMEN
Screening combinatorial libraries of conformationally constrained peptides against macromolecular targets is utilized in identifying novel drug leads and in developing new reagents for chemical biology. In methods such as phage-display selections, biotinylated macromolecular targets are often immobilized on avidin- and streptavidin-functionalized supports. Thus, the characterization of peptides that bind avidin and streptavidin is necessary for accurate interpretation of screening and selection results. Toward this goal, we panned a phage-displayed cyclic peptide library against NeutrAvidin, a chemically deglycosylated version of avidin. The selection produced a highly homologous consensus motif (Asp-Arg/Leu-Ala-Ser/Thr-Pro-Tyr/Trp). Two of these cyclic peptides, CDRATPYC and CDRASPYC, bound both NeutrAvidin and avidin with low-micromolar dissociation constants, whereas their acyclic counterparts had negligible affinity (< 80-fold). Moreover, these cyclic peptides were very specific for their targets and did not bind the structurally and functionally similar protein, streptavidin. Thus, we have identified a new class of cyclic peptides, distinct from the much-studied streptavidin-binding His-Pro-Gln peptide motif. These results will not only allow for discriminating between desired and background cyclic peptide motifs in selections and screens but also provide a new protein/peptide model system and a useful reagent in chemical biology that can have utility in protein immobilization, purification, and chemical tagging.
Asunto(s)
Avidina/química , Biblioteca de Péptidos , Péptidos Cíclicos/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Avidina/síntesis química , Avidina/metabolismo , Compuestos Azo/metabolismo , Bacteriófago M13/química , Bacteriófago M13/metabolismo , Sitios de Unión , Diseño de Fármacos , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos Cíclicos/síntesis química , Péptidos Cíclicos/metabolismo , Conformación Proteica , Estreptavidina/química , Estreptavidina/metabolismoRESUMEN
New approaches for identifying small molecules that specifically target protein surfaces as opposed to active site clefts are of much current interest. Toward this goal, we describe a three-step methodology: in step one, we target a protein of interest by directed evolution of a small beta-sheet scaffold; in step two, we identify residues on the scaffold that are implicated in binding; and in step three, we transfer the chemical information from the beta-sheet to a small molecule mimic. As a case study, we targeted the proteolytic enzyme thrombin, involved in blood coagulation, utilizing a library of beta-sheet epitopes displayed on phage that were previously selected for conservation of structure. We found that the thrombin-binding, beta-sheet displaying mini-proteins retained their structure and stability while inhibiting thrombin at low micromolar inhibition constants. A conserved dityrosine recognition motif separated by 9.2 A was found to be common among the mini-protein inhibitors and was further verified by alanine scanning. A molecule containing two tyrosine residues separated by a linker that matched the spacing on the beta-sheet scaffold inhibited thrombin, whereas a similar dityrosine molecule separated by a shorter 6 A linker could not. Moreover, kinetic analysis revealed that both the mini-protein as well as its minimalist mimic with only two functional residues exhibited noncompetitive inhibition of thrombin. Thus, this reductionist approach affords a simple methodology for transferring information from structured protein scaffolds to yield small molecule leads for targeting protein surfaces with novel mechanisms of action.
Asunto(s)
Epítopos/química , Estructura Secundaria de Proteína , Proteínas/química , Secuencia de Aminoácidos , Sitios de Unión , Inmunoglobulina G/química , Inmunoglobulina G/metabolismo , Cinética , Datos de Secuencia Molecular , Proteínas/metabolismo , Trombina/antagonistas & inhibidores , Trombina/química , Trombina/metabolismo , Tirosina/químicaRESUMEN
We describe the directed evolution of a miniature beta-sheet protein for targeting beta-amyloid oligomers implicated in Alzheimer's disease. Circular dichroism spectroscopy, thermal denaturation experiments, and immunoglobulin binding assays established that our beta-amyloid-targeted miniature protein, TJ10, presents a well-folded thermostable beta-sheet. TJ10 was found to prevent beta-amyloid fibrillization at stoichiometric concentrations and was also an effective inhibitor at substoichiometric concentrations. Thus our results provide a new and potent beta-sheet chemical template for effectively targeting beta-amyloid while also demonstrating a general strategy for targeting proteins implicated in other amyloidogenic diseases.
Asunto(s)
Péptidos beta-Amiloides/antagonistas & inhibidores , Secuencia de Aminoácidos , Péptidos beta-Amiloides/química , Dicroismo Circular , Microscopía Electrónica de Transmisión , Datos de Secuencia MolecularRESUMEN
We have characterized two homologous, single-point core mutants of a 57-residue, hyperthermophilic variant of the B1 domain of protein G (HTB1). These single-point mutations in HTB1 replace a Phe residue in the hydrophobic core with either a Glu or Asp residue. Both of these homologous core-variant mutants undergo significant structural rearrangement from the native, monomeric fold and exist as stable soluble oligomeric species of 5 and 30 nm in diameter. Gel-filtration, dynamic light scattering, circular dichroism spectroscopy, fluorescence spectroscopy, along with Congo Red and Thioflavin T binding clearly demonstrated that these core-variants undergo significant structural rearrangement from the native, monomeric ubiquitin fold. The two oligomeric species did not equilibrate over extended periods of time and displayed distinct secondary structures. The larger of the two species was found to possess structural features that are reminiscent of an emerging class of protein assemblies prone to beta-sheet-mediated aggregation. These results are significant as there are very few examples of extensive conformational or oligomerization switching brought about by single-point mutations in a stable protein-fold.