Toward the assembly and characterization of an encoded library hit confirmation platform: Bead-Assisted Ligand Isolation Mass Spectrometry (BALI-MS).

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.

Selecting Approaches for Hit Identification and Increasing Options by Building the Efficient Discovery of Actionable Chemical Matter from DNA-Encoded Libraries.

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.

Ultra-High-Throughput Acoustic Droplet Ejection-Open Port Interface-Mass Spectrometry for Parallel Medicinal Chemistry.

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.

Photoredox cross-electrophile coupling in DNA-encoded chemistry.

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.

Photocatalytic [2 + 2] Cycloaddition in DNA-Encoded Chemistry.

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.

Merging C(sp3)-H activation with DNA-encoding.

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.

Detection and Removal of Small Molecule and Endotoxin Contaminants in ADC Preparations.

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.

A Solution Phase Platform to Characterize Chemical Reaction Compatibility with DNA-Encoded Chemical Library Synthesis.

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.

Natural Product Bis-Intercalator Depsipeptides as a New Class of Payloads for Antibody-Drug Conjugates.

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.

Kinetically guided radical-based synthesis of C(sp3)-C(sp3) linkages on DNA.

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.

Cytotoxic Spliceostatins from Burkholderia sp. and Their Semisynthetic Analogues.

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.

Spliceostatin hemiketal biosynthesis in Burkholderia spp. is catalyzed by an iron/α-ketoglutarate-dependent dioxygenase.

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.

Evaluating indole-related derivatives as precursors in the directed biosynthesis of diazepinomicin analogues.

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.

Probing natural product biosynthetic pathways using Fourier transform ion cyclotron resonance mass spectrometry.

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.

Isolation, structure elucidation, and synthesis of eudistomides A and B, lipopeptides from a Fijian ascidian Eudistoma sp.

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.

Investigating the biosynthetic origin of the nitro group in pyrrolomycins.

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.

Theopapuamide, a cyclic depsipeptide from a Papua New Guinea lithistid sponge Theonella swinhoei.

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.

Chemical transformation of prostaglandin-A2: a novel series of C-10 halogenated, C-12 hydroxylated prostaglandin-A2 analogues.

[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.

Aurantosides G, H, and I: three new tetramic acid glycosides from a Papua New Guinea Theonella swinhoei.

Aurantosides G-I (1-3) have been isolated from the lithistid sponge Theonella swinhoei from Papua New Guinea. Their structures were established by spectroscopic and chemical methods. Compounds 1-3 represent new monochloropentaenoyl tetramic acids with mono-, di-, and tri-N-saccharide substituents, respectively. Aurantosides G-I (1-3) failed to show any significant cytotoxicity against the human colon tumor cell line HCT-116.

Olefin cross-metathesis as a tool in natural product degradation. The stereochemistry of (+)-falcarindiol.

[reaction: see text] There are conflicting reports in the literature concerning the absolute sterochemistry at C-3 of the common plant polyacetylene oxylipin (+)-falcarindiol. We have employed olefin cross-metathesis using Grubbs' second generation catalyst and ethylene gas to degrade falcarindiol to the symmetrical 1,9-decadiene-4,6-diyne-3,8-diol. The reaction is completely selective for net removal of the aliphatic side chain. Degradation of (+)-falcarindiol from Tetraplasandra hawaiiensis yields a meso product as shown by chiral HPLC. Hence, (+)-falcarindiol from this source has a (3R,8S)-configuration.