High-Throughput DNA-Encoded Libraries Affinity Selection Platform for Binder Identification with Solid Support Protein Immobilization.

DNA-encoded libraries (DELs) have demonstrated to be one of the most powerful technologies within the ligand identification toolbox, widely used either in academia or biotech and pharma companies. DEL methodology utilizes affinity selection (AS) as the approach to interrogate the protein of interest for the identification of binders. Here we present a high-throughput, fully automated AS platform developed to fulfill industrial standards and compatible with different assay formats to improve the reproducibility of the AS process for DEL binders identification. This platform is flexible enough to virtually set aside all kinds of DELs and AS methods and conditions using immobilized proteins. It bears the two main immobilization methods to support of the proteins of interest: magnetic beads or resin tip columns. A combination of a broad variety of protocol options with a wide range of different experimental conditions can be set up with a throughput of 96 samples at the same time. In addition, small modifications of the protocols provide the platform with the versatility to run not only the routine DEL screens, but also test covalent libraries, the successful immobilization of the proteins of interest, and many other experiments that may be required. This versatile AS platform for DEL can be a powerful instrument for direct application of the technology in academic and industry settings.

Design, synthesis and validation of a new Crimped Head-Piece for DNA-Encoded libraries generation.

Codification of DNA Encoded Libraries (DELs) is critical for successful ligand identification of molecules that bind a protein of interest (POI). There are different encoding strategies that permit, for instance, the customization of a DEL for testing single or dual pharmacophores (single strand DNA) or for producing and screening large diversity libraries of small molecules (double strand DNA). Both approaches challenges, either from the synthetic and encoding point of view, or from the selection methodology to be utilized for the screening. The Head-Piece contains the DNA sequence that is attached to a chemical compound, allowing the encoding of each molecule with a unique DNA tag. Designing the Head-Piece for a DNA-encoded library involves careful consideration of several key aspects including DNA barcode identity, sequence length and attachment chemistry. Here we describe a double stranded DNA versatile Head-Piece that can be used for the generation of single or dual pharmacophore libraries, but also shows other advanced DEL functionalities, stability and enlarged encoding capacity.

Normalization of DNA encoded library affinity selection results driven by high throughput sequencing and HPLC purification.

The output of an affinity selection screening results in a huge amount of valuable data that, after conducting the appropriate analysis, lead to the correct identification of the compounds enriched in the target of interest. The approach chosen to perform these analyses has become a key step in the development of a successful DNA Encoded Library platform. In this paper, we describe the combination of High Performance Liquid Chromatography purification during the library production with the Next Generation Sequencing analysis of the libraries to assess the yield of the chemical reactions prior to the affinity selection. This process allows us, apart from achieving higher quality libraries, to enable a normalization analysis of the affinity selection output, thus minimizing the bias induced by the chemical yield of each reaction as a misleading factor within the analysis and subsequent compound short-listing for off-DNA synthesis.

On-DNA Palladium-Catalyzed Hydrogenation-like Reaction Suitable for DNA-Encoded Library Synthesis.

Herein we describe a method to orthogonally remove on-DNA N-Cbz, N-Alloc, N-Allyl, O-Bn, and O-Allyl protecting groups in the presence of other common protecting groups to afford free amines and carboxylic acids, respectively. The developed method uses NaBH4 as the source of hydrogen in the presence of Pd(OAc)2 under DNA aqueous conditions. In addition, under the developed conditions we were able to successfully hydrogenate triple and double bonds to totally saturated compounds. Furthermore, we introduce a new alternative procedure to reduce azides and aromatic nitro compounds to primary amines.

Mild and Efficient Palladium-Mediated C-N Cross-Coupling Reaction between DNA-Conjugated Aryl Bromides and Aromatic Amines.

DNA-encoded library technology (ELT) has emerged in the pharmaceutical industry as a powerful tool for hit and lead generation. Over the last 10 years, a number of DNA-compatible chemical reactions have been published and used to synthesize libraries. Among the most commonly used reactions in medicinal chemistry is the C-N bond formation, and its application to DNA-encoded library technology affords an alternative approach to identify high-affinity binders for biologically relevant protein targets. Herein we report a newly developed Pd-promoted C-N cross coupling reaction between DNA-conjugated aryl bromides and a wide scope of arylamines in good to excellent yields. The mild reaction conditions should facilitate the synthesis of novel DNA-encoded combinatorial libraries.

Design and Development of a Technology Platform for DNA-Encoded Library Production and Affinity Selection.

DNA-encoded libraries (DELs) have emerged as an efficient and cost-effective drug discovery tool for the exploration and screening of very large chemical space using small-molecule collections of unprecedented size. Herein, we report an integrated automation and informatics system designed to enhance the quality, efficiency, and throughput of the production and affinity selection of these libraries. The platform is governed by software developed according to a database-centric architecture to ensure data consistency, integrity, and availability. Through its versatile protocol management functionalities, this application captures the wide diversity of experimental processes involved with DEL technology, keeps track of working protocols in the database, and uses them to command robotic liquid handlers for the synthesis of libraries. This approach provides full traceability of building-blocks and DNA tags in each split-and-pool cycle. Affinity selection experiments and high-throughput sequencing reads are also captured in the database, and the results are automatically deconvoluted and visualized in customizable representations. Researchers can compare results of different experiments and use machine learning methods to discover patterns in data. As of this writing, the platform has been validated through the generation and affinity selection of various libraries, and it has become the cornerstone of the DEL production effort at Lilly.

tagFinder: A Novel Tag Analysis Methodology That Enables Detection of Molecules from DNA-Encoded Chemical Libraries.

Available tools to analyze sequencing data coming from DNA-encoded chemical libraries (DELs) are often limited to in-house methods, which usually rely on strictly looking for the particular DEL structure used. Current methods do not take into account technological errors, such as library codification and sequencing errors, when detecting the sequences. The vast amount of data produced by next-generation sequencing of DEL screens is usually enough to extract the minimum information needed for compound identification. Here, we report a methodology to deconvolute encoding oligonucleotides, thus optimizing the sequencing power regardless of the library size, design complexity, or sequencing technology chosen. tagFinder is a highly flexible tool for fast tag detection and thorough DEL results characterization, which requires minimal hardware resources, scales linearly, and does not introduce any analytical error. The methodology can even deal with sequencing errors and PCR duplicates on single- or double-stranded DNA, enhancing the analytical detection and quantification of molecules and the informativeness of the entire process. Source code is available at https://github.com/jamigo/tagFinder .

Autologous Stromal Vascular Fraction in the Intravenous Treatment of End-Stage Chronic Obstructive Pulmonary Disease: A Phase I Trial of Safety and Tolerability.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a consistently progressive, ultimately fatal disease for which no treatment exists capable of either reversing or even interrupting its course. It afflicts more than 5% of the population in many countries, and it accordingly represents the third most frequent cause of death in the US, where it accounts for more than 600 billion in health care costs, morbidity, and mortality. Adipose tissue contains within its stromal compartment a high abundance of adipose stem/stromal cells (ASCs), which can be readily separated from the adipocyte population by methods which require less than 2 h of processing time and yield a concentrated cellular preparation termed the stromal vascular fraction (SVF). The SVF contains all cellular elements of fat, excluding adipocytes. Recent clinical studies have begun to explore the feasibility and safety of the local injection or intravascular delivery of SVF or more purified populations of ASCs derived by culture protocols. Several pre-clinical studies have demonstrated a remarkable ability of ASC to nearly fully ameliorate the progress of emphysema due to cigarette smoke exposure as well as other causes. However, no prior clinical studies have evaluated the safety of administration of either ASC or SVF in subjects with COPD. We hypothesized that harvest, isolation, and immediate intravenous infusion of autologous SVF would be feasible and safe in subjects with COPD; and that such an approach, if ultimately determined to be efficacious as well as safe, would provide a highly practical method for treatment of COPD. METHODS: In this study, an initial phase I trial evaluating the early and delayed safety of SVF infusion was performed. Twelve subjects were enrolled in the study, in which adipose tissue was harvested using standard liposuction techniques, followed by SVF isolation and intravenous infusion of 150 - 300 million cells. Standardized questionnaires were administered to study feasibility as well as immediate and delayed outcomes and adverse events as primary endpoints. Secondary endpoints included subjective wellness and attitudes towards the procedure, as well as willingness to undergo the procedure a second time. The follow-up time ranged from 3 to 12 months, averaging 12 months. RESULTS: Of the 12 subjects, only one experienced an immediate adverse event, related to bruising from the liposuction. No observed pulmonary or cardiac issues were observed as related to the procedure. There were no deaths over the 12-month study period, and none identified in the subsequent telephonic follow-up. Attitudes toward the procedure were predominantly positive, and 92% of the study subjects expressed a desire to undergo the procedure a second time. CONCLUSIONS: This study is the first to demonstrate safety of SVF infusion in humans with serious pulmonary disease. Specifically, the use of intravenous infusion as a route to achieve pulmonary cellular targeting did not lead to clinical pulmonary compromise. The intravenous administration of SVF should be further explored as a potentially feasible and safe method for delivery leading to possible therapeutic benefit.

Structural characterization and quantitation of compound loading of disubstituted benzoates bound to Wang resin through high-resolution magic angle spinning NMR spectroscopy.

The combination of 1D and 2D high-resolution magic angle spinning NMR experiments led to the assignment of the proton and carbon resonances for several disubstituted benzoates bound to a polystyrene resin through a Wang linker. It is shown that the signal corresponding to the methylene protons of the linker can be utilized to monitor the solid-phase reactions and determine the loading of the compounds on the resin.

Reconstrucción de extremidades inferiores con colgajo sural diferido / Sural flap in the coverage of lower extremity wounds using differed transposition technique

Objetivo: Evaluar la viabilidad del colgajo sural, en lesiones de extremidades inferiores que comprometen la zona de su pedículo vascular utilizando la técncia quirúrgica de transposición diferida o de retardo. Materiales y métodos: Se estudiaron 08 pacientes sometidos a cirugía reconstructiva con colgajo sural diferido por lesiones en tercio distal de extremidades inferiores que comprometían la zona de su pedículo vascular, desde junio 2001 a mayo 2003, en el Servicio de Cirugía Plástica del Hospital Nacional Cayetano Heredia. Resultados: El 100 por ciento de colgajos estuvo viable al momento de realizar la transposición. Las complicaciones encontradas fueron menores, siendo la más frecuente la necrosis parcial. Conclusiones: La técnica diferida utilizada en el colgajo sural, cuando la lesión compromete la zona de su pedículo vascular, es adecuada para mantener la viabilidad del colgajo.

Evaluación del Elisa VIH como prueba única en la detección de infectados VIH asintomáticos en población cerrada: Años 1992-1999 / Evaluation of Elisa-VIH as a unique test in the detection of asymptomatic patients infected by VIH in a close population: 1992-1999

Objetivo: El propósito de este estudio fue determinar la estrategia a usar con los modernos test de Elisa-VIH, a fin de prescindir del Western blot como test confirmativo, en razón de disminuir costos. Métodos: Estudio prospectivo de la incidencia de verdaderos positivos y negativos del test Elisa-VIH en población cerrada centro referencial a nivel Fuerza Aérea del Perú, en los años 1992 al 1999. La prevalencia fue obtenida en los años 1992 y 1993. Se obtuvo el valor predictivo positivo y negativo entre los años 1994 a 1999. Nuestra técnica fue similar a la estrategia II de Sato para prevalencias menores de 1 por ciento pero se diferencia en que se corren dos Elisa-VIH con la primera muestra, si sale reactivo el primer resultado. Resultado: Se realizó 37 791 test serológicos en población con prevalencia global de 0.1 por ciento. La prevalencia fue considerada como una constante a través del año. Se obtuvo un valor predictivo positivo del 100 por ciento y valor predictivo negativo del 100 por ciento. Conclusiones: Se concluye que no es necesario Realizar el test de Western blot si se aplica la técnica propuesta para casos de vigilancia en población con prevalencia menor de 1 por ciento.