RESUMEN
The ability to predict chemical structure from DNA sequence has to date been a necessary cornerstone of DNA-encoded library technology. DNA-encoded libraries (DELs) are typically screened by immobilized affinity selection and enriched library members are identified by counting the number of times an individual compound's sequence is observed in the resultant dataset. Those with high signal reads (DEL hits) are subsequently followed up through off-DNA synthesis of the predicted small molecule structures. However, hits followed-up in this manner often fail to translate to confirmed ligands. To address this low conversion rate of DEL hits to off-DNA ligands, we have developed an approach that eliminates the reliance on chemical structure prediction from DNA sequence. Here we describe our method of combining non-combinatorial resynthesis on-DNA following library procedures as a rapid means to assess the probable molecules attached to the DNA barcode. Furthermore, we apply our Bead-Assisted Ligand Isolation Mass Spectrometry (BALI-MS) technique to identify the true binders found within the mixtures of on-DNA synthesis products. Finally, we describe a Normalized Enrichment (NE) metric that allows for the quantitative assessment of affinity selection in these studies. We exemplify how this combined approach enables the identification of putative hit matter against a clinically relevant therapeutic target bisphosphoglycerate mutase, BPGM.
Asunto(s)
ADN/química , Descubrimiento de Drogas , Biblioteca de Genes , Espectrometría de Masas/métodos , Técnicas Químicas Combinatorias , Ligandos , Estructura Molecular , Bibliotecas de Moléculas Pequeñas/químicaRESUMEN
Over the past 20 years, the toolbox for discovering small-molecule therapeutic starting points has expanded considerably. Pharmaceutical researchers can now choose from technologies that, in addition to traditional high-throughput knowledge-based and diversity screening, now include the screening of fragment and fragment-like libraries, affinity selection mass spectrometry, and selection against DNA-encoded libraries (DELs). Each of these techniques has its own unique combination of advantages and limitations that makes them more, or less, suitable for different target classes or discovery objectives, such as desired mechanism of action. Layered on top of this are the constraints of the drug-hunters themselves, including budgets, timelines, and available platform capacity; each of these can play a part in dictating the hit identification strategy for a discovery program. In this article, we discuss some of the factors that we use to govern our building of a hit identification roadmap for a program and describe the increasing role that DELs are playing in our discovery strategy. Furthermore, we share our learning during our initial exploration of DEL and highlight the approaches we have evolved to maximize the value returned from DEL selections. Topics addressed include the optimization of library design and production, reagent validation, data analysis, and hit confirmation. We describe how our thinking in these areas has led us to build a DEL platform that has begun to deliver tractable matter to our global discovery portfolio.
Asunto(s)
Descubrimiento de Drogas/métodos , Biblioteca de Genes , Bibliotecas de Moléculas Pequeñas , Descubrimiento de Drogas/normas , HumanosRESUMEN
High-throughput experimentation (HTE) has emerged as an important tool in drug discovery, providing a platform for preparing large compound libraries and enabling swift reaction screening over wide-ranging conditions. Recent advances in automated high-density, material-sparing HTE have necessitated the development of rapid analytics with sensitivity and resolution sufficient to identify products and/or assess reaction performance in a timely and data-rich manner. Combination of an ultrathroughput (UT) reader platform with Acoustic Droplet Ejection-Open Port Interface-Mass Spectrometry (ADE-OPI-MS) provides the requisite speed and sensitivity. Herein, we report the application of ADE-OPI-MS to HTE in the areas of parallel medicinal chemistry and reaction screening.
RESUMEN
A catalytic manifold that enables photoredox cross-electrophile coupling of alkyl bromides with DNA-tagged aryl iodides in aqueous solution is presented. This metallaphotoredox transformation was aided by the identification of a new pyridyl bis(carboxamidine) ligand, which proved critical to the nickel catalytic cycle. The described C(sp2)-C(sp3) coupling tolerates a wide range of both DNA-tagged aryl iodides as well as alkyl bromides. Importantly, this reaction was optimized for parallel synthesis, which is a paramount prerequisite for the preparation of combinatorial libraries, by using a 96-well plate-compatible blue LED array as the light source. Therefore, this mild and DNA-compatible transformation is well positioned for the construction of DNA-encoded libraries.
Asunto(s)
Alcanos/química , Bromuros/química , ADN/química , Hidrocarburos Aromáticos/química , Yoduros/química , Alcanos/síntesis química , Alquilación , Bromuros/síntesis química , Catálisis , Técnicas Químicas Combinatorias , ADN/síntesis química , Hidrocarburos Aromáticos/síntesis química , Yoduros/síntesis química , Ligandos , Níquel/química , Oxidación-Reducción , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/químicaRESUMEN
The on-DNA synthesis of highly substituted cyclobutanes was achieved through a photocatalytic [2 + 2] cycloaddition reaction in aqueous solution. Readily available DNA-tagged styrene derivatives were reacted with structurally diverse cinnamates in the presence of an iridium-based photocatalyst, Ir(ppy)2(dtbbpy)PF6, to forge two new C(sp3)-C(sp3) bonds. This transformation was demonstrated to have excellent functional group tolerance and allowed for the facile installation of a variety of heteroaromatic substituents on a densely functionalized cyclobutane scaffold.
Asunto(s)
Ciclobutanos/química , ADN/síntesis química , Catálisis , Reacción de Cicloadición , ADN/química , Estructura Molecular , Procesos FotoquímicosRESUMEN
DNA-encoded library (DEL) technology has the potential to dramatically expedite hit identification in drug discovery owing to its ability to perform protein affinity selection with millions or billions of molecules in a few experiments. To expand the molecular diversity of DEL, it is critical to develop different types of DNA-encoded transformations that produce billions of molecules with distinct molecular scaffolds. Sequential functionalization of multiple C-H bonds provides a unique avenue for creating diversity and complexity from simple starting materials. However, the use of water as solvent, the presence of DNA, and the extremely low concentration of DNA-encoded coupling partners (0.001 M) have hampered the development of DNA-encoded C(sp3)-H activation reactions. Herein, we report the realization of palladium-catalyzed C(sp3)-H arylation of aliphatic carboxylic acids, amides and ketones with DNA-encoded aryl iodides in water. Notably, the present method enables the use of alternative sets of monofunctional building blocks, providing a linchpin to facilitate further setup for DELs. Furthermore, the C-H arylation chemistry enabled the on-DNA synthesis of structurally-diverse scaffolds containing enriched C(sp3) character, chiral centers, cyclopropane, cyclobutane, and heterocycles.
RESUMEN
Incomplete removal of free (unconjugated) drug or drug-linker species used to prepare ADCs results in contaminated ADC samples which may pose a risk for toxicity. Due to the extreme potency of typical small molecule toxins employed in ADCs, even relatively low levels of free drug contaminants in ADC samples have been hypothesized to result in nonspecific (i.e., off-target) activity in biological systems. It is possible for trace levels of certain free drug species to persist in final ADC samples despite the inclusion of common purification steps during the preparation processes. Therefore, methods for the detection, quantification, and removal of residual free drug present in ADC samples are ultimately required for the preparation of safe and efficacious final ADC drug products. Herein we report general methods for the detection and removal of such contaminants.
Asunto(s)
Contaminación de Medicamentos , Endotoxinas/química , Endotoxinas/aislamiento & purificación , Inmunoconjugados/química , Inmunoconjugados/aislamiento & purificación , Cromatografía Líquida de Alta Presión/métodos , Cromatografía Líquida de Alta Presión/normas , Endotoxinas/análisis , Humanos , Inmunoconjugados/análisis , Sensibilidad y EspecificidadRESUMEN
DNA-encoded chemical library (DECL) synthesis must occur in aqueous media under conditions that preserve the integrity of the DNA encoding tag. While the identification of "DNA-compatible" reaction conditions is critical for the development of DECL designs that explore previously inaccessible chemical space, reports measuring such compatibility have been largely restricted to methods that do not faithfully capture the impact of reaction conditions on DNA fidelity in solution phase. Here we report a comprehensive methodology that uses soluble DNA substrates that exactly recapitulate DNA's exposure to the chemically reactive species of DECL synthesis. This approach includes the assessment of chemical fidelity (reaction yield and purity), encoding fidelity (ligation efficiency), and readability (DNA compatibility), revealing the fate of the DNA tag during DECL chemistry from a single platform.
Asunto(s)
ADN/química , Bibliotecas de Moléculas Pequeñas/síntesis química , Técnicas Químicas Combinatorias , Estructura Molecular , Bibliotecas de Moléculas Pequeñas/química , SolucionesRESUMEN
A potent class of DNA-damaging agents, natural product bis-intercalator depsipeptides (NPBIDs), was evaluated as ultrapotent payloads for use in antibody-drug conjugates (ADCs). Detailed investigation of potency (both in cells and via biophysical characterization of DNA binding), chemical tractability, and in vitro and in vivo stability of the compounds in this class eliminated a number of potential candidates, greatly reducing the complexity and resources required for conjugate preparation and evaluation. This effort yielded a potent, stable, and efficacious ADC, PF-06888667, consisting of the bis-intercalator, SW-163D, conjugated via an N-acetyl-lysine-valine-citrulline- p-aminobenzyl alcohol- N, N-dimethylethylenediamine (AcLysValCit-PABC-DMAE) linker to an engineered variant of the anti-Her2 mAb, trastuzumab, catalyzed by transglutaminase.
Asunto(s)
Productos Biológicos/química , Depsipéptidos/química , Inmunoconjugados/química , Sustancias Intercalantes/química , Animales , Antineoplásicos Inmunológicos/química , Línea Celular Tumoral , ADN/química , Depsipéptidos/sangre , Depsipéptidos/farmacocinética , Equinomicina/química , Genes erbB-2 , Semivida , Xenoinjertos , Humanos , Ratones , Trastuzumab/químicaRESUMEN
DNA-encoded libraries (DEL)-based discovery platforms have recently been widely adopted in the pharmaceutical industry, mainly due to their powerful diversity and incredible number of molecules. In the two decades since their disclosure, great strides have been made to expand the toolbox of reaction modes that are compatible with the idiosyncratic aqueous, dilute, and DNA-sensitive parameters of this system. However, construction of highly important C(sp3)-C(sp3) linkages on DNA through cross-coupling remains unexplored. In this article, we describe a systematic approach to translating standard organic reactions to a DEL setting through the tactical combination of kinetic analysis and empirical screening with information captured from data mining. To exemplify this model, implementation of the Giese addition to forge high value C-C bonds on DNA was studied, which represents a radical-based synthesis in DEL.
Asunto(s)
ADN/química , Biblioteca de Genes , Modelos Moleculares , CinéticaRESUMEN
As part of our efforts to develop new classes of tubulin inhibitor payloads for antibody-drug conjugate (ADC) programs, we developed a tubulysin ADC that demonstrated excellent in vitro activity but suffered from rapid metabolism of a critical acetate ester. A two-pronged strategy was employed to address this metabolism. First, the hydrolytically labile ester was replaced by a carbamate functional group resulting in a more stable ADC that retained potency in cellular assays. Second, site-specific conjugation was employed in order to design ADCs with reduced metabolic liabilities. Using the later approach, we were able to identify a conjugate at the 334C position of the heavy chain that resulted in an ADC with considerably reduced metabolism and improved efficacy. The examples discussed herein provide one of the clearest demonstrations to-date that site of conjugation can play a critical role in addressing metabolic and PK liabilities of an ADC. Moreover, a clear correlation was identified between the hydrophobicity of an ADC and its susceptibility to metabolic enzymes. Importantly, this study demonstrates that traditional medicinal chemistry strategies can be effectively applied to ADC programs.
RESUMEN
Spliceostatins are potent spliceosome inhibitors biosynthesized by a hybrid nonribosomal peptide synthetase-polyketide synthase (NRPS-PKS) system of the trans-acyl transferase (AT) type. Burkholderia sp. FERM BP-3421 produces hemiketal spliceostatins, such as FR901464, as well as analogs containing a terminal carboxylic acid. We provide genetic and biochemical evidence for hemiketal biosynthesis by oxidative decarboxylation rather than the previously hypothesized Baeyer-Villiger oxidative release postulated to be catalyzed by a flavin-dependent monooxygenase (FMO) activity internal to the last module of the PKS. Inactivation of Fe(II)/α-ketoglutarate-dependent dioxygenase gene fr9P led to loss of hemiketal congeners, whereas the mutant was still able to produce all major carboxylic acid-type compounds. FMO mutants, on the other hand, produced both hemiketal and carboxylic acid analogs containing an exocyclic methylene instead of an epoxide, indicating that the FMO is involved in epoxidation rather than Baeyer-Villiger oxidation. Moreover, recombinant Fr9P enzyme was shown to catalyze hydroxylation to form ß-hydroxy acids, which upon decarboxylation led to hemiketal FR901464. Finally, a third oxygenase activity encoded in the biosynthetic gene cluster, the cytochrome P450 monooxygenase Fr9R, was assigned as a 4-hydroxylase based on gene inactivation results. Identification and deletion of the gene involved in hemiketal formation allowed us to generate a strain--the dioxygenase fr9P(-) mutant--that accumulates only the carboxylic acid-type spliceostatins, which are as potent as the hemiketal analogs, when derivatized to increase cell permeability, but are chemically more stable.
Asunto(s)
Burkholderia/metabolismo , Dioxigenasas/metabolismo , Hierro/metabolismo , Biocatálisis , Burkholderia/enzimología , Datos de Secuencia MolecularRESUMEN
The spliceostatin class of natural products was reported to be potent cytotoxic agents via inhibition of the spliceosome, a key protein complex in the biosynthesis of mature mRNA. As part of an effort to discover novel leads for cancer chemotherapy, we re-examined this class of compounds from several angles, including fermentation of the producing strains, isolation and structure determination of new analogues, and semisynthetic modification. Accordingly, a group of spliceostatins were isolated from a culture broth of Burkholderia sp. FERM BP-3421, and their structures identified by analysis of spectroscopic data. Semisynthesis was performed on the major components 4 and 5 to generate ester and amide derivatives with improved in vitro potency. With their potent activity against tumor cells and unique mode of action, spliceostatins can be considered potential leads for development of cancer drugs.
Asunto(s)
Antineoplásicos/aislamiento & purificación , Antineoplásicos/farmacología , Burkholderia/química , Piranos/aislamiento & purificación , Piranos/farmacología , Compuestos de Espiro/aislamiento & purificación , Compuestos de Espiro/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/química , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Estructura Molecular , Piranos/síntesis química , Piranos/química , ARN Mensajero/biosíntesis , Compuestos de Espiro/síntesis química , Compuestos de Espiro/química , Relación Estructura-ActividadRESUMEN
The effectiveness of precursor-directed biosynthesis to generate diazepinomicin (1) analogues with varied ring-A substitutents was investigated by feeding commercially available, potential ring-A precursors such as fluorinated tryptophans, halogenated anthranilates, and various substituted indoles into growing actinomycete culture DPJ15 (genus Micromonospora). Two new monofluorinated diazepinomicin analogues (2 and 3) were identified and characterized by spectroscopic methods. Both derivatives showed modest antibacterial activity against the Gram-positive coccus Staphylococcus aureus with MIC values in the range 8-32 microg/mL.
Asunto(s)
Dibenzazepinas/aislamiento & purificación , Hidrocarburos Fluorados/aislamiento & purificación , Indoles/aislamiento & purificación , Micromonospora/química , Dibenzazepinas/química , Dibenzazepinas/metabolismo , Dibenzazepinas/farmacología , Hidrocarburos Fluorados/química , Hidrocarburos Fluorados/metabolismo , Hidrocarburos Fluorados/farmacología , Indoles/química , Indoles/metabolismo , Indoles/farmacología , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Staphylococcus aureus/efectos de los fármacosRESUMEN
Eudistomides A (1) and B (2), two new cyclic peptides, were isolated from a Fijian ascidian Eudistoma sp. These five-residue cystine-linked cyclic peptides are flanked by a C-terminal methyl ester and a 12-oxo- or 12-hydroxy-tetradecanoyl moiety. The complete structures of the eudistomides were determined using a combination of spectroscopic and chemical methods. Chiral HPLC analysis revealed that all five amino acid residues in 1 and 2 had the L-configuration. Total synthesis of eudistomides A (1) and B (2) confirmed the proposed structures. Enantioselective lipase-catalyzed hydrolysis of a mixture of C-35 acetoxy epimers indicated a 35R absolute configuration for 2.
Asunto(s)
Lipopéptidos/química , Péptidos Cíclicos/química , Urocordados/química , Animales , Cromatografía Líquida de Alta Presión , Lipopéptidos/aislamiento & purificación , Resonancia Magnética Nuclear Biomolecular , Péptidos Cíclicos/aislamiento & purificación , Análisis de Secuencia de Proteína , Espectrometría de Masas en TándemRESUMEN
Two natural products, diazepinomicin (1) and dioxapyrrolomycin (2), containing stable isotopic labels of (15)N or deuterium, were used to demonstrate the utility of Fourier transform ion cyclotron resonance mass spectrometry for probing natural product biosynthetic pathways. The isotopic fine structures of significant ions were resolved and subsequently assigned elemental compositions on the basis of highly accurate mass measurements. In most instances the mass measurement accuracy is less than one part per million (ppm), which typically makes the identification of stable-isotope labeling unambiguous. In the case of the mono-(15)N-labeled diazepinomicin (1) derived from labeled tryptophan, tandem mass spectrometry located this (15)N label at the non-amide nitrogen. Through the use of exceptionally high mass resolving power of over 125,000, the isotopic fine structure of the molecular ion cluster of 1 was revealed. Separation of the (15)N(2) peak from the isobaric (13)C(15)N peak, both having similar abundances, demonstrated the presence of a minor amount of doubly (15)N-labeled diazepinomicin (1). Tandem mass spectrometry amplified this isotopic fine structure (Deltam=6.32 mDa) from mDa to 1 Da scale thereby allowing more detailed scrutiny of labeling content and location. Tandem mass spectrometry was also used to assign the location of deuterium labeling in two deuterium-labeled diazepinomicin (1) samples. In one case three deuterium atoms were incorporated into the dibenzodiazepine core; while in the other a mono-D label was mainly incorporated into the farnesyl side chain. The specificity of (15)N-labeling in dioxapyrrolomycin (2) and the proportion of the (15)N-label contained in the nitro group were determined from the measurement of the relative abundance of the (14)NO(2)(1-) and (15)NO(2)(1-) fragment ions.
Asunto(s)
Productos Biológicos/biosíntesis , Ciclotrones , Dibenzazepinas/metabolismo , Espectrometría de Masas en Tándem/métodos , Deuterio , Fermentación , Análisis de Fourier , Pirroles/metabolismoRESUMEN
Feasible modes of introducing the nitro group into pyrrolomycin antibiotics were investigated based on incorporation of (15)N-labeled arginine and proline into dioxapyrrolomycin, produced by the actinomycete culture LL-F42248. Biosynthesis of nitrated pyrrolomycins was unaffected by the presence of nitric oxide synthase (NOS) inhibitors. The culture was able to grow in nitrogen-free (minimal) media and produce nitrated secondary metabolites. These results indicate that LL-F42248 is capable of fixing nitrogen.
Asunto(s)
Actinomyces/metabolismo , Óxido Nítrico Sintasa/antagonistas & inhibidores , Fijación del Nitrógeno/fisiología , Arginina/metabolismo , Arginina/farmacología , Atmósfera/química , Estructura Molecular , Nitrógeno/metabolismo , Pirroles/química , Pirroles/farmacología , omega-N-Metilarginina/metabolismo , omega-N-Metilarginina/farmacologíaRESUMEN
Theopapuamide (1), a new cytotoxic peptide, has been isolated from the lithistid sponge Theonella swinhoei from Papua New Guinea. The structure was established by analysis of NMR, mass spectrometry, and chemical methods. The undecapeptide (1) contains several unusual amino acid residues, of which the occurrence of beta-methoxyasparagine and 4-amino-5-methyl-2,3,5-trihydroxyhexanoic acid (Amtha) is unprecedented in natural peptides. Compound 1 also contains an amide-linked fatty acid moiety, 3-hydroxy-2,4,6-trimethyloctanoic acid (Htoa). Theopapuamide (1) was cytotoxic against CEM-TART and HCT-116 cell lines, with EC50 values of 0.5 and 0.9 microM, respectively.
Asunto(s)
Antineoplásicos/aislamiento & purificación , Depsipéptidos/aislamiento & purificación , Theonella/química , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Depsipéptidos/química , Depsipéptidos/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Papúa Nueva GuineaRESUMEN
Mutations in the adenomatous polyposis coli (APC) tumor suppressor gene seem to underlie the initiation of many colorectal carcinomas. Loss of APC function results in accumulation of beta-catenin and activation of beta-catenin/TCF-dependent transcription. Recent studies have implicated APC in controlling retinoic acid biosynthesis during normal intestinal development through a WNT-independent mechanism. Paradoxically, however, previous studies found that dietary supplementation of Apc(MIN) mice with retinoic acid failed to abrogate adenoma formation. While investigating the above finding, we found that expression of CYP26A1, a major retinoic acid catabolic enzyme, was up-regulated in Apc(MIN) mouse adenomas, human FAP adenomas, human sporadic colon carcinomas, and in the intestine of apc(mcr) mutant zebrafish embryos. Mechanistically, cyp26a1 induction following apc mutation is dependent on WNT signaling as antisense morpholino knockdown of tcf4 or injection of a dnLEF construct into apc(mcr) mutant zebrafish suppressed expression of cyp26a1 along with known WNT target genes. In addition, injection of stabilized beta-catenin or dnGSK3beta into wild-type embryos induced cyp26a1 expression. Genetic knockdown or pharmacologic inhibition of cyp26a1 in apc(mcr) mutant zebrafish embryos rescued gut differentiation defects such as expression of intestinal fatty acid-binding protein and pancreatic trypsin. These findings support a novel role for APC in balancing retinoic acid biosynthesis and catabolism through WNT-independent and WNT-dependent mechanisms.
Asunto(s)
Proteína de la Poliposis Adenomatosa del Colon/deficiencia , Neoplasias del Colon/metabolismo , Sistema Enzimático del Citocromo P-450/biosíntesis , Intestinos/patología , Proteínas Wnt/metabolismo , Proteína de la Poliposis Adenomatosa del Colon/genética , Animales , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Neoplasias del Colon/enzimología , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Inhibidores Enzimáticos del Citocromo P-450 , Sistema Enzimático del Citocromo P-450/genética , Regulación Enzimológica de la Expresión Génica , Humanos , Intestinos/efectos de los fármacos , Intestinos/enzimología , Ratones , Morfolinas/farmacología , Oligonucleótidos/genética , Oligonucleótidos/farmacología , Ácido Retinoico 4-Hidroxilasa , Transducción de Señal , Tretinoina/metabolismo , Tretinoina/farmacología , Regulación hacia Arriba , Pez Cebra , Proteínas de Pez CebraRESUMEN
[reaction: see text] Synthesis of a novel class of C-10 halogenated and C-12 oxygenated prostaglandin-A(2) derivatives (6a-6c) has been accomplished. (15S)-Prostaglandin-A(2) (1), from the gorgonian Plexaura homomalla, served as the starting material for the synthesis. The absolute configuration was determined using NMR.