A chemical ligation method for construction of DNA-encoded small-molecule libraries has been developed. Taking advantage of the ability of the Klenow fragment of DNA polymerase to accept templates with triazole linkages in place of phosphodiesters, we have designed a strategy for chemically ligating oligonucleotide tags using cycloaddition chemistry. We have utilized this strategy in the construction and selection of a small molecule library, and successfully identified inhibitors of the enzyme soluble epoxide hydrolase.
A vast number of biological processes are mediated by multivalent ligand-receptor interactions, including cell adhesion, host invasion by pathogens, pathogen neutralization by host and numerous cell regulatory signaling pathways. [1] Multivalency is especially important for carbohydrate-receptor interactions: whereas individual glycans [2] may bind with low affinity to a single binding site, the clustering of glycans creates a high-avidity interaction with clustered binding sites. This "carbohydrate cluster effect" [1b] has been demonstrated experimentally with synthetic multivalent carbohydrate ligands which bind well to protein targets. These ligands have included oligo-and polyvalent clusters of glycans on diverse scaffolds, including small molecules, dendrimers, polymers and even viral capsids.To date, most glycocluster ligands have been designed for synthetic convenience rather than control of tertiary structure. However, the biological activity of the natural glycocluster may be influenced by tertiary structure and other elements which are not usually addressed in synthetic glycocluster designs, such as: 1) Glycan spacing and orientation -glycans are normally attached to synthetic scaffolds through long flexible linkers, and the scaffolds themselves are often flexible. [3] 2) Glycan internal flexibility -in a natural glycocluster, As an alternative to rational design, we have been interested in directed evolution-based design of glycocluster ligands. Figure 1 outlines this concept: a library of scaffold molecules is glycosylated, generating a library of glycoclusters. The "best" glycoclusters are selected from the pool by binding to the target protein. These selection winners are then replicated to form a second-generation library and the process is repeated for several rounds until the pool is sufficiently enriched in high-affinity binders. We have chosen DNA as our glycocluster scaffolding material because DNA is easy to synthesize, easy to replicate by PCR, can fold into diverse sequence-dependent structures, and is amenable to sequence-specific "glycosylation" by glycan azides using CuAAC [5] ("click") attachment to alkyne-modified nucleobases. Iterative selection/amplification of DNA structures (SELEX) is often performed to obtain DNAs which bind to a target. [6] Our method, by contrast, would yield DNA scaffolds whose major function would be to position and support glycans optimally for target binding. However, these DNAs might also contain elements which would interact directly with the target, mimicking any non-carbohydrate components necessary in the natural ligand.We decided to test this concept in the design of glycoclusters which mimic the epitope of 2G12, an antibody which protects against HIV infection and binds to a cluster of highmannose glycans on the HIV envelope protein gp120. [7] Rationally-designed clusters of these glycans have been tested as vaccines to elicit 2G12-like antibodies, but without success. [8] Our evolution-based design would be the product of the procedure outlined i...
Millions of individuals are infected with and die from tuberculosis (TB) each year, and multidrug-resistant (MDR) strains of TB are increasingly prevalent. As such, there is an urgent need to identify novel drugs to treat TB infections. Current frontline therapies include the drug isoniazid, which inhibits the essential NADH-dependent enoyl–acyl-carrier protein (ACP) reductase, InhA. To inhibit InhA, isoniazid must be activated by the catalase-peroxidase KatG. Isoniazid resistance is linked primarily to mutations in the katG gene. Discovery of InhA inhibitors that do not require KatG activation is crucial to combat MDR TB. Multiple discovery efforts have been made against InhA in recent years. Until recently, despite achieving high potency against the enzyme, these efforts have been thwarted by lack of cellular activity. We describe here the use of DNA-encoded X-Chem (DEX) screening, combined with selection of appropriate physical properties, to identify multiple classes of InhA inhibitors with cell-based activity. The utilization of DEX screening allowed the interrogation of very large compound libraries (1011 unique small molecules) against multiple forms of the InhA enzyme in a multiplexed format. Comparison of the enriched library members across various screening conditions allowed the identification of cofactor-specific inhibitors of InhA that do not require activation by KatG, many of which had bactericidal activity in cell-based assays.
We have identified and characterized novel potent inhibitors of Bruton's tyrosine kinase (BTK) from a single DNA-encoded library of over 110 million compounds by using multiple parallel selection conditions, including variation in target concentration and addition of known binders to provide competition information. Distinct binding profiles were observed by comparing enrichments of library building block combinations under these conditions; one enriched only at high concentrations of BTK and was competitive with ATP, and another enriched at both high and low concentrations of BTK and was not competitive with ATP. A compound representing the latter profile showed low nanomolar potency in biochemical and cellular BTK assays. Results from kinetic mechanism of action studies were consistent with the selection profiles. Analysis of the co-crystal structure of the most potent compound demonstrated a novel binding mode that revealed a new pocket in BTK. Our results demonstrate that profile-based selection strategies using DNA-encoded libraries form the basis of a new methodology to rapidly identify small molecule inhibitors with novel binding modes to clinically relevant targets.
Mcl-1 is a pro-apoptotic BH3 protein family member similar to Bcl-2 and Bcl-xL. Overexpression of Mcl-1 is often seen in various tumors and allows cancer cells to evade apoptosis. Here we report the discovery and optimization of a series of non-natural peptide Mcl-1 inhibitors. Screening of DNA-encoded libraries resulted in hit compound , a 1.5 μM Mcl-1 inhibitor. A subsequent crystal structure demonstrated that compound bound to Mcl-1 in a β-turn conformation, such that the two ends of the peptide were close together. This proximity allowed for the linking of the two ends of the peptide to form a macrocycle. Macrocyclization resulted in an approximately 10-fold improvement in binding potency. Further exploration of a key hydrophobic interaction with Mcl-1 protein and also with the moiety that engages Arg256 led to additional potency improvements. The use of protein-ligand crystal structures and binding kinetics contributed to the design and understanding of the potency gains. Optimized compound is a<3 nM Mcl-1 inhibitor, while inhibiting Bcl-2 at only 5 μM and Bcl-xL at >99 μM, and induces cleaved caspase-3 in MV4-11 cells with an IC of 3 μM after 6 h.
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