Gene/Protein Disease Symptom Drug Enzyme Compound
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Small Molecule Microarrays (SMMs) represent a general platform for screening small molecule-protein interactions independent of functional inhibition of target proteins. In an effort to increase the scope and utility of SMMs, we have modified the SMM screening methodology to increase assay sensitivity and facilitate multiplex screening. Fusing target proteins to the HaloTag protein allows us to covalently prelabel fusion proteins with fluorophores, leading to increased assay sensitivity and an ability to conduct multiplex screens. We use the interaction between FKBP12 and two ligands, rapamycin and ARIAD's "bump" ligand, to show that the HaloTag-based SMM screening methodology significantly increases assay sensitivity. Additionally, using wild type FKBP12 and the FKBP12 F36V mutant, we show that prelabeling various protein isoforms with different fluorophores allows us to conduct multiplex screens and identify ligands to a specific isoform. Finally, we show this multiplex screening technique is capable of identifying ligands selective for a specific PTP1B isoform using a 20,000 compound screening deck.
ACS Chem Biol 2012 Dec 21
PMID:A HaloTag-based small molecule microarray screening methodology with increased sensitivity and multiplex capabilities. 2301 33

In drug discovery, small molecules must often discriminate between healthy and diseased cells. This feat is usually accomplished by binding to a protein that is preferentially expressed in the target cell or on its surface. However, in many cases, the expression of an individual protein may not generate sufficient cyto-selectivity. Here, we demonstrate that bispecific molecules can better discriminate between similar cell types by exploiting their simultaneous affinity for two proteins. Inspired by the natural product FK506, we designed molecules that have affinity for both FKBP12 and HIV protease. Using cell-based reporters and live virus assays, we observed that these compounds preferentially accumulated in cells that express both targets, mimicking an infected lymphocyte. Treatment with FKBP12 inhibitors reversed this partitioning, while overexpression of FKBP12 protein further promoted it. The partitioning into the target cell type could be tuned by controlling the properties of the linker and the affinities for the two proteins. These results show that bispecific molecules create significantly better potential for cyto-selectivity, which might be especially important in the development of safe and effective antivirals and anticancer compounds.
ACS Chem Biol 2015 Nov 20
PMID:Selective Targeting of Cells via Bispecific Molecules That Exploit Coexpression of Two Intracellular Proteins. 2632 64

Transcriptional control can be used to program cells to label proteins with noncanonical amino acids by regulating the expression of orthogonal aminoacyl tRNA synthetases (aaRSs). However, we cannot yet program cells to control labeling in response to aaRS and ligand binding. To identify aaRSs whose activities can be regulated by interactions with ligands, we used a combinatorial approach to discover fragmented variants of Escherichia coli methionyl tRNA synthetase (MetRS) that require fusion to associating proteins for maximal activity. We found that these split proteins could be leveraged to create ligand-dependent MetRS using two approaches. When a pair of MetRS fragments was fused to FKBP12 and the FKBP-rapamycin binding domain (FRB) of mTOR and mutations were introduced that direct substrate specificity toward azidonorleucine (Anl), Anl metabolic labeling was significantly enhanced in growth medium containing rapamycin, which stabilizes the FKBP12-FRB complex. In addition, fusion of MetRS fragments to the termini of the ligand-binding domain of the estrogen receptor yielded proteins whose Anl metabolic labeling was significantly enhanced when 4-hydroxytamoxifen (4-HT) was added to the growth medium. These findings suggest that split MetRS can be fused to a range of ligand-binding proteins to create aaRSs whose metabolic labeling activities depend upon post-translational interactions with ligands.
ACS Synth Biol 2017 08 18
PMID:Programming Post-Translational Control over the Metabolic Labeling of Cellular Proteins with a Noncanonical Amino Acid. 2841 2

FKBP12 ligands such as FK506 have been shown to activate the BMP signaling pathway and facilitate tissue regeneration. However, the immunosuppressive activity of FK506 limits its clinical application. Using Heck reaction, we generated nonimmunosuppressive analogs of FK506 by fusing heterocycles to the calcineurin (CN) binding domain of FK506. Structure-activity relationships provided novel mechanistic insights into the FK506-CN interaction that can be exploited for rational design of future analogs.
ACS Med Chem Lett 2019 Sep 12
PMID:One-step Heck Reaction Generates Nonimmunosuppressive FK506 Analogs for Pharmacological BMP Activation. 3153 Nov 97

On the basis of computational design, a focused one-bead one-compound library has been prepared on microparticle-encoded PEGA1900 beads consisting of small tripeptides with a triazole-capped N-terminal. The library was screened towards a double point-mutated version of the human FKBP12 protein, known as the destabilizing domain (DD). Inspired by the decoded library hits, unnatural peptide structures were screened in a novel on-bead assay, which was useful for a rapid structure evaluation prior to off-bead resynthesis. Subsequently, a series of 19 compounds were prepared and tested using a competitive fluorescence polarization assay, which led to the discovery of peptide ligands with low micromolar binding affinity towards the DD. The methodology represents a rapid approach for identification of a novel structure scaffold, where the screening and initial structure refinement was accomplished using small quantities of library building blocks.
ACS Comb Sci 2020 03 09
PMID:Design and Combinatorial Development of Shield-1 Peptide Mimetics Binding to Destabilized FKBP12. 3202 20

Monitoring microbial reactions in highly opaque or autofluorescent environments like soils, seawater, and wastewater remains challenging. To develop a simple approach for observing post-translational reactions within microbes situated in environmental matrices, we designed a methyl halide transferase (MHT) fragment complementation assay that reports by synthesizing an indicator gas. We show that backbone fission within regions of high sequence variability in the Rossmann domain yields split MHT (sMHT) AND gates whose fragments cooperatively associate to synthesize CH3Br. Additionally, we identify a sMHT whose fragments require fusion to pairs of interacting partner proteins for maximal activity. We also show that sMHT fragments fused to FKBP12 and the FKBP-rapamycin binding domain of mTOR display significantly enhanced CH3Br production in the presence of rapamycin. This gas production is reversed in the presence of the competitive inhibitor of FKBP12/FKPB dimerization, indicating that sMHT is a reversible reporter of post-translational reactions. This sMHT represents the first genetic AND gate that reports on protein-protein interactions via an indicator gas. Because indicator gases can be measured in the headspaces of complex environmental samples, this assay should be useful for monitoring the dynamics of diverse molecular interactions within microbes situated in hard-to-image marine and terrestrial matrices.
ACS Synth Biol 2020 11 20
PMID:A Split Methyl Halide Transferase AND Gate That Reports by Synthesizing an Indicator Gas. 3310 25