13 research outputs found
DNA-Encoded Solid-Phase Synthesis: Encoding Language Design and Complex Oligomer Library Synthesis
The
promise of exploiting combinatorial synthesis for small molecule
discovery remains unfulfilled due primarily to the “structure
elucidation problem”: the back-end mass spectrometric analysis
that significantly restricts one-bead-one-compound (OBOC) library
complexity. The very molecular features that confer binding potency
and specificity, such as stereochemistry, regiochemistry, and scaffold
rigidity, are conspicuously absent from most libraries because isomerism
introduces mass redundancy and diverse scaffolds yield uninterpretable
MS fragmentation. Here we present DNA-encoded solid-phase synthesis
(DESPS), comprising parallel compound synthesis in organic solvent
and aqueous enzymatic ligation of unprotected encoding dsDNA oligonucleotides.
Computational encoding language design yielded 148 thermodynamically
optimized sequences with Hamming string distance ≥ 3 and total
read length <100 bases for facile sequencing. Ligation is efficient
(70% yield), specific, and directional over 6 encoding positions.
A series of isomers served as a testbed for DESPS’s utility
in split-and-pool diversification. Single-bead quantitative PCR detected
9 Ă— 10<sup>4</sup> molecules/bead and sequencing allowed for
elucidation of each compound’s synthetic history. We applied
DESPS to the combinatorial synthesis of a 75 645-member OBOC
library containing scaffold, stereochemical and regiochemical diversity
using mixed-scale resin (160-ÎĽm quality control beads and 10-ÎĽm
screening beads). Tandem DNA sequencing/MALDI-TOF MS analysis of 19
quality control beads showed excellent agreement (<1 ppt) between
DNA sequence-predicted mass and the observed mass. DESPS synergistically
unites the advantages of solid-phase synthesis and DNA encoding, enabling
single-bead structural elucidation of complex compounds and synthesis
using reactions normally considered incompatible with unprotected
DNA. The widespread availability of inexpensive oligonucleotide synthesis,
enzymes, DNA sequencing, and PCR make implementation of DESPS straightforward,
and may prompt the chemistry community to revisit the synthesis of
more complex and diverse libraries
Reprogramming Urokinase into an Antibody-Recruiting Anticancer Agent
Synthetic compounds for controlling or creating human
immunity
have the potential to revolutionize disease treatment. Motivated by
challenges in this arena, we report herein a strategy to target metastatic
cancer cells for immune-mediated destruction by targeting the urokinase-type
plasminogen activator receptor (uPAR). Urokinase-type plasminogen
activator (uPA) and uPAR are overexpressed on the surfaces of a wide
range of invasive cancer cells and are believed to contribute substantially
to the migratory propensities of these cells. The key component of
our approach is an <u>a</u>ntibody-<u>r</u>ecruiting <u>m</u>olecule that targets the <u>u</u>rokinase receptor (ARM-U). This bifunctional construct
is formed by selectively, covalently attaching an antibody-binding
small molecule to the active site of the urokinase enzyme. We demonstrate
that ARM-U is capable of directing antibodies to the surfaces of target
cancer cells and mediating both antibody-dependent cellular phagocytosis
(ADCP) and antibody-dependent cellular cytotoxicity (ADCC) against
multiple human cancer cell lines. We believe that the reported strategy
has the potential to inform novel treatment options for a variety
of deadly, invasive cancers
Chemically Synthesized Molecules with the Targeting and Effector Functions of Antibodies
This article reports the design,
synthesis, and evaluation of a
novel class of molecules of intermediate size (approximately 7000
Da), which possess both the targeting and effector functions of antibodies.
These compoundsî—¸called synthetic antibody mimics targeting
prostate cancer (SyAM-Ps)î—¸bind simultaneously to prostate-specific
membrane antigen and Fc gamma receptor I, thus eliciting highly selective
cancer cell phagocytosis. SyAMs have the potential to combine the
advantages of both small-molecule and biologic therapies, and may
address many drawbacks associated with available treatments for cancer
and other diseases
High-throughput identification of DNA-encoded IgG ligands that distinguish active and latent Mycobacterium tuberculosis infections
The circulating antibody repertoire encodes a patient's health status and pathogen exposure history, but identifying antibodies with diagnostic potential usually requires knowledge of the antigen(s). We previously circumvented this problem by screening libraries of bead-displayed small molecules against case and control serum samples to discover "epitope surrogates" (Ligands of IgGs enriched in the case sample). Here, we describe an improved version of this technology that employs DNA-encoded libraries and high-throughput FACS-based screening to discover epitope surrogates that differentiate noninfectious/latent (LTB) patients from infectious/active TB (ATB) patients, which is imperative for proper treatment selection and antibiotic stewardship. Normal control/LTB (10 patients each, NCL) and ATB (10 patients) serum pools were screened against a library (5 Ă— 10 beads, 448 000 unique compounds) using fluorescent antihuman IgG to label hit compound beads for FACS. Deep sequencing decoded all hit structures and each hit's occurrence frequencies. ATB hits were pruned of NCL hits and prioritized for resynthesis based on occurrence and homology. Several structurally homologous families were identified and 16/21 resynthesized representative hits validated as selective ligands of ATB serum IgGs (p < 0.005). The native secreted TB protein Ag85B (though not the E. coli recombinant form) competed with one of the validated ligands for binding to antibodies, suggesting that it mimics a native Ag85B epitope. The use of DNA-encoded libraries and FACS-based screening in epitope surrogate discovery reveals thousands of potential hit structures. Distilling this list down to several consensus chemical structures yielded a diagnostic panel for ATB composed of thermally stable and economically produced small molecule ligands in place of protein antigens