RESUMEN
We report the discovery of drug-like small molecules that bind specifically to the precursor of the oncogenic and pro-inflammatory microRNA-21 with mid-nanomolar affinity. The small molecules target a local structure at the Dicer cleavage site and induce distinctive structural changes in the RNA, which correlate with specific inhibition of miRNA processing. Structurally conservative single nucleotide substitutions eliminate the conformational change induced by the small molecules, which is also not observed in other miRNA precursors. The most potent of these compounds reduces cellular proliferation and miR-21 levels in cancer cell lines without inhibiting kinases or classical receptors, while closely related compounds without this specific binding activity are inactive in cells. These molecules are highly ligand-efficient (MW < 330) and display specific biochemical and cellular activity by suppressing the maturation of miR-21, thereby providing an avenue toward therapeutic development in multiple diseases where miR-21 is abnormally expressed.
Asunto(s)
MicroARNs , MicroARNs/metabolismo , Línea CelularRESUMEN
The microRNAs are non-coding RNAs which post-transcriptionally regulate the expression of many eukaryotic genes, and whose dysregulation is a driver of human disease. Here we report the discovery of a very slow (0.1 s-1) conformational rearrangement at the Dicer cleavage site of pre-miR-21, which regulates the relative concentration of readily- and inefficiently-processed RNA structural states. We show that this dynamic switch is affected by single nucleotide mutations and can be biased by small molecule and peptide ligands, which can direct the microRNA to occupy the inefficiently processed state and reduce processing efficiency. This result reveals a new mechanism of RNA regulation and suggests a chemical approach to suppressing or activating pathogenic microRNAs by selective stabilization of their unprocessed or processed states.
Asunto(s)
MicroARNs , Procesamiento Postranscripcional del ARN , Estabilidad del ARN , Riboswitch , Humanos , Ligandos , MicroARNs/química , Conformación de Ácido Nucleico , División del ARN , Ribonucleasa III/químicaRESUMEN
We describe a scalable nuclear magnetic resonance (NMR) screening approach to identify and prioritize small molecule fragments that bind to structured RNAs. This approach is target agnostic and, therefore, amenable to many RNA structures and libraries, and it provides initial hits for further synthetic elaboration and structure-based drug discovery efforts. We demonstrate the approach on the pre-miR-21 stem-loop, which is of significant interest in oncology and metabolic diseases. We screened the pre-miR-21 hairpin using a small (420 compounds) commercially available fragment library and identified 18 hits in the first round of triage screening. This was further refined to four fragments which passed all screening cascade filters. Among these four hits, a thiadiazole fragment was demonstrated to bind the Dicer cleavage site of pre-miR-21 by target-detected NMR experiments and through the observation of clear intermolecular NOEs.
RESUMEN
Specialized translation initiation is a novel form of regulation of protein synthesis, whereby RNA structures within the 5'-UTR regulate translation rates of specific mRNAs. Similar to internal ribosome entry sites (IRESs), specialized translation initiation requires the recruitment of eukaryotic initiation factor 3 (eIF3), but also requires cap recognition by eIF3d, a new 5'-m7GTP recognizing protein. How these RNA structures mediate eIF3 recruitment to affect translation of specific mRNAs remains unclear. Here, we report the nuclear magnetic resonance (NMR) structure of a stem-loop within the c-JUN 5' UTR recognized by eIF3 and essential for specialized translation initiation of this well-known oncogene. The structure exhibits similarity to eIF3 recognizing motifs found in hepatitis C virus (HCV)-like IRESs, suggesting mechanistic similarities. This work establishes the RNA structural features involved in c-JUN specialized translation initiation and provides a basis to search for small molecule inhibitors of aberrant expression of the proto-oncogenic c-JUN.
Asunto(s)
Regiones no Traducidas 5' , Factor 3 de Iniciación Eucariótica/metabolismo , Sitios Internos de Entrada al Ribosoma , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Biosíntesis de Proteínas , ARN/química , Ribosomas/metabolismo , Factor 3 de Iniciación Eucariótica/genética , Hepacivirus/química , Hepacivirus/genética , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/genética , Conformación de Ácido Nucleico , Iniciación de la Cadena Peptídica Traduccional , Unión Proteica , ARN/genética , ARN/metabolismo , Ribosomas/genéticaRESUMEN
The HIV-1 trans-activator protein Tat binds the trans-activation response element (TAR) to facilitate recruitment of the super elongation complex (SEC) to enhance transcription of the integrated pro-viral genome. The Tat-TAR interaction is critical for viral replication and the emergence of the virus from the latent state, therefore, inhibiting this interaction has long been pursued to discover new anti-viral or latency reversal agents. However, discovering active compounds that directly target RNA with high affinity and selectivity remains a significant challenge; limiting pre-clinical development. Here, we report the rational design of a macrocyclic peptide mimic of the arginine rich motif of Tat, which binds to TAR with low pM affinity and 100-fold selectivity against closely homologous RNAs. Despite these unprecedented binding properties, the new ligand (JB181) only moderately inhibits Tat-dependent reactivation in cells and recruitment of positive transcription elongation factor (P-TEFb) to TAR. The NMR structure of the JB181-TAR complex revealed that the ligand induces a structure in the TAR loop that closely mimics the P-TEFb/Tat1:57/AFF4/TAR complex. These results strongly suggest that high-affinity ligands which bind the UCU bulge are not likely to inhibit recruitment of the SEC and suggest that targeting of the TAR loop will be an essential feature of effective Tat inhibitors.
Asunto(s)
Infecciones por VIH/genética , Duplicado del Terminal Largo de VIH/genética , VIH-1/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Antivirales/química , Antivirales/farmacología , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/virología , Duplicado del Terminal Largo de VIH/efectos de los fármacos , VIH-1/efectos de los fármacos , VIH-1/patogenicidad , Humanos , Ligandos , Complejos Multiproteicos/efectos de los fármacos , Complejos Multiproteicos/genética , Factor B de Elongación Transcripcional Positiva/química , Factor B de Elongación Transcripcional Positiva/genética , Unión Proteica , ARN Viral/genética , Transcripción Genética/efectos de los fármacos , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/químicaRESUMEN
The RNA recognition motif (RRM), which is the most abundant RNA-binding motif in eukaryotes, is a well-structured domain of about 90 amino acids, yet the ß2ß3 hairpin, corresponding to strands 2 and 3 of the ß-sheet, and the intervening loop make essential interactions with RNA in many RRM complexes. A series of small cyclic peptide mimics of the ß2ß3 hairpin of Rbfox2 protein that recognize the terminal loop of precursor miR-20b have been designed to investigate whether the full RNA-binding protein can be mimicked with a minimal structurally preorganized peptide. Within a small library of seven cyclic peptides, a peptide with low-micromolar affinity for the miR-20b precursor was found. NMR spectroscopy titration data suggest that this peptide specifically targets the apical loop of pre-miR-20b. This work shows that it is possible to mimic RNA-binding proteins with designed stable peptides, which provide a starting point for designing or evolving small peptide mimetics of RRM proteins.
Asunto(s)
MicroARNs/metabolismo , Péptidos Cíclicos/metabolismo , Peptidomiméticos/metabolismo , Factores de Empalme de ARN/química , Proteínas Represoras/química , Secuencia de Aminoácidos , Humanos , Péptidos Cíclicos/química , Peptidomiméticos/química , Prueba de Estudio Conceptual , Unión Proteica , Dominios Proteicos , Motivo de Reconocimiento de ARNRESUMEN
Mixed-chirality peptide macrocycles such as cyclosporine are among the most potent therapeutics identified to date, but there is currently no way to systematically search the structural space spanned by such compounds. Natural proteins do not provide a useful guide: Peptide macrocycles lack regular secondary structures and hydrophobic cores, and can contain local structures not accessible with l-amino acids. Here, we enumerate the stable structures that can be adopted by macrocyclic peptides composed of l- and d-amino acids by near-exhaustive backbone sampling followed by sequence design and energy landscape calculations. We identify more than 200 designs predicted to fold into single stable structures, many times more than the number of currently available unbound peptide macrocycle structures. Nuclear magnetic resonance structures of 9 of 12 designed 7- to 10-residue macrocycles, and three 11- to 14-residue bicyclic designs, are close to the computational models. Our results provide a nearly complete coverage of the rich space of structures possible for short peptide macrocycles and vastly increase the available starting scaffolds for both rational drug design and library selection methods.
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Simulación por Computador , Diseño Asistido por Computadora , Modelos Químicos , Péptidos/química , Estabilidad Proteica , Diseño de Fármacos , Resonancia Magnética Nuclear Biomolecular , Pliegue de ProteínaRESUMEN
MicroRNAs (miRNAs) help orchestrate cellular growth and survival through post-transcriptional mechanisms. The dysregulation of miRNA biogenesis can lead to cellular growth defects and chemotherapeutic resistance and plays a direct role in the development of many chronic diseases. Among these RNAs, miR-21 is consistently overexpressed in most human cancers, leading to the down-regulation of key tumor-suppressing and pro-apoptotic factors, suggesting that inhibition of miR-21 biogenesis could reverse these negative effects. However, targeted inhibition of miR-21 using small molecules has had limited success. To overcome difficulties in targeting RNA secondary structure with small molecules, we developed a class of cyclic ß-hairpin peptidomimetics which bind to RNA stem-loop structures, such as miRNA precursors, with potent affinity and specificity. We screened an existing cyclic peptide library and discovered a lead structure which binds to pre-miR21 with KD = 200 nM and prefers it over other pre-miRNAs. The NMR structure of the complex shows that the peptide recognizes the Dicer cleavage site and alters processing of the precursor to the mature miRNA in vitro and in cultured cells. The structure provides a rationale for the peptide binding activity and clear guidance for further improvements in affinity and targeting.
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Anticarcinógenos/química , MicroARNs/antagonistas & inhibidores , Péptidos Cíclicos/farmacología , Peptidomiméticos/farmacología , Animales , Línea Celular , ARN Helicasas DEAD-box/antagonistas & inhibidores , ARN Helicasas DEAD-box/metabolismo , Humanos , Ligandos , MicroARNs/metabolismo , Biblioteca de Péptidos , Péptidos Cíclicos/metabolismo , Peptidomiméticos/metabolismo , Unión Proteica , Procesamiento Postranscripcional del ARN , Ribonucleasa III/antagonistas & inhibidores , Ribonucleasa III/metabolismo , Especificidad por SustratoRESUMEN
The interface between the DnaG primase C-terminal domain (CTD) and the N-terminal domain of DnaB helicase is essential for bacterial DNA replication because it allows coordinated priming of DNA synthesis at the replication fork while the DNA is being unwound. Because these two proteins are conserved in all bacteria and distinct from those in eukaryotes, their interface is an attractive antibiotic target. To learn more about this interface, we determined the solution structure and dynamics of the DnaG primase CTD from Staphylococcus aureus, a medically important bacterial species. Comparison with the known primase CTD structures shows there are two biologically relevant conformations, an open conformation that likely binds to DnaB helicase and a closed conformation that does not. The S. aureus primase CTD is in the closed conformation, but nuclear magnetic resonance (NMR) dynamic studies indicate there is considerable movement in the linker between the two subdomains and that N564 is the most dynamic residue within the linker. A high-throughput NMR ligand affinity screen identified potential binding compounds, among which were acycloguanosine and myricetin. Although the affinity for these compounds and adenosine was in the millimolar range, all three bind to a common pocket that is present only on the closed conformation of the CTD. This binding pocket is at the opposite end of helices 6 and 7 from N564, the key hinge residue. The identification of this binding pocket should allow the development of stronger-binding ligands that can prevent formation of the CTD open conformation that binds to DnaB helicase.
Asunto(s)
ADN Primasa/química , ADN Primasa/metabolismo , Staphylococcus aureus/enzimología , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Sitios de Unión , Ligandos , Modelos Moleculares , Simulación del Acoplamiento Molecular , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Dominios ProteicosRESUMEN
Small molecules that bind to RNA potently and specifically are relatively rare. The study of molecules that bind to the HIV-1 transactivation response (TAR) hairpin, a cis-acting HIV genomic element, has long been an important model system for the chemistry of targeting RNA. Here we report the synthesis, biochemical, and structural evaluation of a series of molecules that bind to HIV-1 TAR RNA. A promising analogue, 15, retained the TAR binding affinity of the initial hit and displaced a Tat-derived peptide with an IC50 of 40 µM. NMR characterization of a soluble analogue, 2, revealed a noncanonical binding mode for this class of compounds. Finally, evaluation of 2 and 15 by selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) indicates specificity in binding to TAR within the context of an in vitro-synthesized 365-nt HIV-1 5'-untranslated region (UTR). Thus, these compounds exhibit a novel and specific mode of interaction with TAR, providing important suggestions for RNA ligand design.
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Duplicado del Terminal Largo de VIH/efectos de los fármacos , ARN Viral/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Sitios de Unión/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Duplicado del Terminal Largo de VIH/genética , Estructura Molecular , ARN Viral/genética , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-Actividad , Activación Transcripcional/efectos de los fármacos , Activación Transcripcional/genéticaRESUMEN
The increasing appreciation of the central role of non-coding RNAs (miRNAs and long non-coding RNAs) in chronic and degenerative human disease makes them attractive therapeutic targets. This would not be unprecedented: the bacterial ribosomal RNA is a mainstay for antibacterial treatment, while the conservation and functional importance of viral RNA regulatory elements has long suggested they would constitute attractive targets for new antivirals. Oligonucleotide-based chemistry has obvious appeals but also considerable pharmacological limitations that are yet to be addressed satisfactorily. Recent studies identifying small molecules targeting non-coding RNAs may provide an alternative approach to oligonucleotide methods. Here we review recent work investigating new structural and chemical principles for targeting RNA with small molecules.
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Vías Biosintéticas/efectos de los fármacos , Descubrimiento de Drogas/métodos , Modelos Moleculares , ARN no Traducido/química , ARN no Traducido/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/metabolismo , Antibacterianos/farmacología , Antivirales/farmacología , MicroARNs/biosíntesis , MicroARNs/química , Estructura Molecular , ARN no Traducido/uso terapéutico , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
The interaction between DnaG primase and DnaB helicase is essential for stimulating primer synthesis during bacterial DNA replication. The interaction occurs between the N-terminal domain of helicase and the C-terminal domain of primase. Here we present the (1)H, (13)C, and (15)N backbone and side-chain resonance assignments for the C-terminal helicase interaction domain of Staphylococcus aureus primase.
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ADN Helicasas/metabolismo , ADN Primasa/química , ADN Primasa/metabolismo , Resonancia Magnética Nuclear Biomolecular , Staphylococcus aureus/enzimología , Estructura Terciaria de Proteína , SolucionesRESUMEN
We report that proteins with the same function bind the same set of small molecules from a standardized chemical library. This observation led to a quantifiable and rapidly adaptable method for protein functional analysis using experimentally derived ligand binding profiles. Ligand binding is measured using a high-throughput NMR ligand affinity screen with a structurally diverse chemical library. The method was demonstrated using a set of 19 proteins with a range of functions. A statistically significant similarity in ligand binding profiles was only observed between the two functionally identical albumins and between the five functionally similar amylases. This new approach is independent of sequence, structure, or evolutionary information and, therefore, extends our ability to analyze and functionally annotate novel genes.
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Ligandos , Anotación de Secuencia Molecular/métodos , Unión Proteica , Proteínas/metabolismo , Proteínas/fisiología , Proteómica/métodos , Amilasas/metabolismo , Humanos , Espectroscopía de Resonancia Magnética/métodos , Albúmina Sérica/metabolismoRESUMEN
Protein sequence space is vast compared to protein fold space. This raises important questions about how structures adapt to evolutionary changes in protein sequences. A growing trend is to regard protein fold space as a continuum rather than a series of discrete structures. From this perspective, homologous protein structures within the same functional classification should reveal a constant rate of structural drift relative to sequence changes. The clusters of orthologous groups (COG) classification system was used to annotate homologous bacterial protein structures in the Protein Data Bank (PDB). The structures and sequences of proteins within each COG were compared against each other to establish their relatedness. As expected, the analysis demonstrates a sharp structural divergence between the bacterial phyla Firmicutes and Proteobacteria. Additionally, each COG had a distinct sequence/structure relationship, indicating that different evolutionary pressures affect the degree of structural divergence. However, our analysis also shows the relative drift rate between sequence identity and structure divergence remains constant.
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Bacterias/química , Bacterias/clasificación , Proteínas Bacterianas/química , Filogenia , Evolución Molecular , Modelos Moleculares , Pliegue de ProteínaRESUMEN
The proliferation of biological databases and the easy access enabled by the Internet is having a beneficial impact on biological sciences and transforming the way research is conducted. There are approximately 1100 molecular biology databases dispersed throughout the Internet. To assist in the functional, structural and evolutionary analysis of the abundant number of novel proteins continually identified from whole-genome sequencing, we introduce the PROFESS (PROtein Function, Evolution, Structure and Sequence) database. Our database is designed to be versatile and expandable and will not confine analysis to a pre-existing set of data relationships. A fundamental component of this approach is the development of an intuitive query system that incorporates a variety of similarity functions capable of generating data relationships not conceived during the creation of the database. The utility of PROFESS is demonstrated by the analysis of the structural drift of homologous proteins and the identification of potential pancreatic cancer therapeutic targets based on the observation of protein-protein interaction networks. Database URL: http://cse.unl.edu/~profess/
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Bases de Datos de Proteínas , Proteínas/genética , Proteínas/fisiología , Secuencia de Aminoácidos , Minería de Datos , Evolución Molecular , Humanos , Internet , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/fisiopatología , Mapeo de Interacción de Proteínas , Proteínas/química , Alineación de Secuencia , Homología Estructural de ProteínaRESUMEN
Large amounts of data from high-throughput metabolomic experiments are commonly visualized using a principal component analysis (PCA) two-dimensional scores plot. The question of the similarity or difference between multiple metabolic states then becomes a question of the degree of overlap between their respective data point clusters in principal component (PC) scores space. A qualitative visual inspection of the clustering pattern in PCA scores plots is a common protocol. This article describes the application of tree diagrams and bootstrapping techniques for an improved quantitative analysis of metabolic PCA data clustering. Our PCAtoTree program creates a distance matrix with 100 bootstrap steps that describes the separation of all clusters in a metabolic data set. Using accepted phylogenetic software, the distance matrix resulting from the various metabolic states is organized into a phylogenetic-like tree format, where bootstrap values 50 indicate a statistically relevant branch separation. PCAtoTree analysis of two previously published data sets demonstrates the improved resolution of metabolic state differences using tree diagrams. In addition, for metabolomic studies of large numbers of different metabolic states, the tree format provides a better description of similarities and differences between each metabolic state. The approach is also tolerant of sample size variations between different metabolic states.
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Metabolómica/métodos , Análisis por Conglomerados , Hongos/efectos de los fármacos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Análisis de Componente Principal , Urato Oxidasa/genética , Urato Oxidasa/metabolismo , Xantinas/farmacologíaRESUMEN
BACKGROUND: Functional similarity is challenging to identify when global sequence and structure similarity is low. Active-sites or functionally relevant regions are evolutionarily more stable relative to the remainder of a protein structure and provide an alternative means to identify potential functional similarity between proteins. We recently developed the FAST-NMR methodology to discover biochemical functions or functional hypotheses of proteins of unknown function by experimentally identifying ligand binding sites. FAST-NMR utilizes our CPASS software and database to assign a function based on a similarity in the structure and sequence of ligand binding sites between proteins of known and unknown function. METHODOLOGY/PRINCIPAL FINDINGS: The PrgI protein from Salmonella typhimurium forms the needle complex in the type III secretion system (T3SS). A FAST-NMR screen identified a similarity between the ligand binding sites of PrgI and the Bcl-2 apoptosis protein Bcl-xL. These ligand binding sites correlate with known protein-protein binding interfaces required for oligomerization. Both proteins form membrane pores through this oligomerization to release effector proteins to stimulate cell death. Structural analysis indicates an overlap between the PrgI structure and the pore forming motif of Bcl-xL. A sequence alignment indicates conservation between the PrgI and Bcl-xL ligand binding sites and pore formation regions. This active-site similarity was then used to verify that chelerythrine, a known Bcl-xL inhibitor, also binds PrgI. CONCLUSIONS/SIGNIFICANCE: A structural and functional relationship between the bacterial T3SS and eukaryotic apoptosis was identified using our FAST-NMR ligand affinity screen in combination with a bioinformatic analysis based on our CPASS program. A similarity between PrgI and Bcl-xL is not readily apparent using traditional global sequence and structure analysis, but was only identified because of conservation in ligand binding sites. These results demonstrate the unique opportunity that ligand-binding sites provide for the identification of functional relationships when global sequence and structural information is limited.
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Apoptosis , Proteínas Bacterianas/química , Proteínas Portadoras/química , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Salmonella typhimurium/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/fisiología , Benzofenantridinas/farmacología , Proteínas Portadoras/fisiología , Dominio Catalítico , Biología Computacional/métodos , Humanos , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Unión Proteica , Homología de Secuencia de Aminoácido , Programas Informáticos , Proteína bcl-X/metabolismoRESUMEN
A multi-step NMR based screening assay is described for identifying and evaluating chemical leads for their ability to bind a target protein. The multi-step NMR assay provides structure-related information while being an integral part of a structure based drug discovery and design program. The fundamental principle of the multi-step NMR assay is to combine distinct 1D and 2D NMR techniques, in such a manner, that the inherent strengths and weakness associated with each technique is complementary to each other in the screen. By taking advantage of the combined strengths of 1D and 2D NMR experiments, it is possible to minimize protein requirements and experiment time and differentiate between non-specific and stoichiometric binders while being able to verify ligand binding, determine a semi-quantitative dissociation constant, identify the ligand binding site and rapidly determine a protein-ligand co-structure. Furthermore, the quality and physical behavior of the ligand is readily evaluated to determine its appropriateness as a chemical lead. The utility of the multi-step NMR assay is demonstrated with the use of PrgI from Salmonella typhimurium and human serum albumin (HSA) as target proteins.
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Descubrimiento de Drogas , Evaluación Preclínica de Medicamentos/métodos , Espectroscopía de Resonancia Magnética/métodos , Proteínas/efectos de los fármacos , Proteínas Bacterianas , Humanos , Ligandos , Unión Proteica , Salmonella typhimurium/química , Albúmina SéricaRESUMEN
Pseudomonas aeruginosa is the prototypical biofilm-forming gram-negative opportunistic human pathogen. P. aeruginosa is causatively associated with nosocomial infections and with cystic fibrosis. Antibiotic resistance in some strains adds to the inherent difficulties that result from biofilm formation when treating P. aeruginosa infections. Transcriptional profiling studies suggest widespread changes in the proteome during quorum sensing and biofilm development. Many of the proteins found to be upregulated during these processes are poorly characterized from a functional standpoint. Here, we report the solution NMR structure of PA1324, a protein of unknown function identified in these studies, and provide a putative biological functional assignment based on the observed prealbumin-like fold and FAST-NMR ligand screening studies. PA1324 is postulated to be involved in the binding and transport of sugars or polysaccharides associated with the peptidoglycan matrix during biofilm formation.
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Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Pseudomonas aeruginosa/química , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Biopelículas , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Genómica , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Polisacáridos Bacterianos/metabolismo , Unión Proteica , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Percepción de Quorum , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Suramina/metabolismo , Regulación hacia ArribaRESUMEN
Many of today's drug discovery programs use high-throughput screening methods that rely on quick evaluations of protein activity to rank potential chemical leads. By monitoring biologically relevant protein-ligand interactions, NMR can provide a means to validate these discovery leads and to optimize the drug discovery process. NMR-based screens typically use a change in chemical shift or line width to detect a protein-ligand interaction. However, the relatively low throughput of current NMR screens and their high demand on sample requirements generally makes it impractical to collect complete binding curves to measure the affinity for each compound in a large and diverse chemical library. As a result, NMR ligand screens are typically limited to identifying candidates that bind to a protein and do not give any estimate of the binding affinity. To address this issue, a methodology has been developed to rank binding affinities for ligands based on NMR screens that use 1D (1)H NMR line-broadening experiments. This method was demonstrated by using it to estimate the dissociation equilibrium constants for twelve ligands with the protein human serum albumin (HSA). The results were found to give good agreement with previous affinities that have been reported for these same ligands with HSA.