Site-directed ligand discovery

Erlanson, Daniel A.; Braisted, Andrew C.; Raphael, D.R.; Randal, Mike; Stroud, Robert M.; Gordon, Eric M.; Wells, James A.

doi:10.1073/pnas.97.17.9367

Cited by 445 publications

(404 citation statements)

References 32 publications

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“…Not only do covalent ligand-protein complexes offer the advantage of an isolable, homogeneous sample for study, but also in this case we were able to access a continuum of cooperativity modes, significantly expanding the breadth of the analysis. Given the ease with which such covalent small molecules can be discovered via tethering, we anticipate the broader use of such ligands in mechanistic studies of conformationally dynamic protein complexes that comprise critical cellular functions (44)(45)(46).…”

Section: Discussionmentioning

confidence: 99%

Dissecting allosteric effects of activator–coactivator complexes using a covalent small molecule ligand

Wang

Lodge

Fierke³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Allosteric binding events play a critical role in the formation and stability of transcriptional activator-coactivator complexes, perhaps in part due to the often intrinsically disordered nature of one or more of the constituent partners. The kinase-inducible domain interacting (KIX) domain of the master coactivator CREB binding protein/ p300 is a conformationally dynamic domain that complexes with transcriptional activators at two discrete binding sites in allosteric communication. The complexation of KIX with the transcriptional activation domain of mixed-lineage leukemia protein leads to an enhancement of binding by the activation domain of CREB (phosphorylated kinase-inducible domain of CREB) to the second site. A transient kinetic analysis of the ternary complex formation aided by small molecule ligands that induce positive or negative cooperative binding reveals that positive cooperativity is largely governed by stabilization of the bound complex as indicated by a decrease in k off . Thus, this suggests the increased binding affinity for the second ligand is not due to an allosteric creation of a more favorable binding interface by the first ligand. This is consistent with data from us and from others indicating that the on rates of conformationally dynamic proteins approach the limits of diffusion. In contrast, negative cooperativity is manifested by alterations in both k on and k off , suggesting stabilization of the binary complex.IDP | protein-protein interaction P rotein-protein interactions (PPIs) underscore all cellular processes, and the mechanistic dissection of PPI networks is thus a high priority (1-4). A particular challenge is defining the mechanism of PPI formation between intrinsically disordered proteins (IDPs) where allosteric changes play a substantive role (5-7). Allosteric communication between binding sites is vital for the proper function of feedback and regulatory circuits in the cell (8, 9). For example, the conformationally dynamic kinase-inducible domain interacting (KIX) domain of the master transcriptional coactivator CREB binding protein (CBP) undergoes a structural shift and stabilization upon binding to a cognate ligand such as the intrinsically disordered transcriptional activation domain (TAD) of mixed-lineage leukemia protein (MLL) (Fig. 1A) (10). The interaction of a second ligand, phosphorylated kinase-inducible domain (pKID) of CREB, is enhanced (up to twofold) by the presence of MLL and several elegant studies have documented allosteric communication between the two binding sites (11-15). However, the relatively modest affinities (micromolar) of the native complexes and the aggregation propensities and the promiscuous binding profiles of the transcriptional activation domains have hampered kinetic dissection of this and other complexes (16,17).The coactivator CBP exists across metazoans (18)(19)(20) and is a transcription hub that interacts with numerous transcriptional activators, using several discrete domains (21,22). The KIX domain within CBP is a 90-residue motif ...

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Section: Discussionmentioning

confidence: 99%

Dissecting allosteric effects of activator–coactivator complexes using a covalent small molecule ligand

Wang

Lodge

Fierke³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…The ligand-binding potential of the hydrophobic and acidic subsites was explored using the tethering method of fragment discovery (Erlanson and Hansen 2004;Erlanson et al 2000). Tethering uses a library of disulfide-exchangeable fragments to select compounds that bind to a site of interest near a native or engineered cysteine residue (Fig.…”

Section: Ligand-binding Potential and Surface Plasticity Of Il-2mentioning

confidence: 99%

“…In fragment-based discovery, a compound half the size of a traditional drug (e.g., 250 Da) is screened for binding to a target of interest. Biophysical approaches are generally favored over functional assays because fragment/protein interactions tend to have low affinity, and binding methods -such as NMR (Shuker et al 1996), X-ray (Carr and Jhoti 2002), surface plasmon resonance (SPR) (Hamalainen et al 2008), or Tethering (Erlanson et al 2000) -provide additional information about binding site and/or binding stoichiometry. Active fragments are then linked or evolved in a second step and retested for binding or inhibition of the target.…”

Section: Introductionmentioning

confidence: 99%

Small-Molecule Inhibitors of IL-2/IL-2R: Lessons Learned and Applied

Wilson

Arkin

2010

Current Topics in Microbiology and Immunology

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“…A related strategy, used for drug discovery screening, is tethering library ligands to a cysteine-mutant receptor by disulfide formation (13,14,15). Here the small molecules are in disulfide form, to react with free cysteine on the protein; a reducing buffer containing a thiol provides selective pressure so that only the ligands with highest affinity remain bound in vitro.…”

Section: Introductionmentioning

confidence: 99%

Cysteinylated Protein as Reactive Disulfide: An Alternative Route to Affinity Labeling

et al. 2007

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Engineering the permanent formation of a receptor-ligand complex has a number of promising applications in chemistry, biology, and medicine. Antibodies and other proteins can be excellent receptors for synthetic ligands such as probes or drugs. Because proteins possess an array of nucleophilic sites, the placement of an electrophile on the synthetic ligand to react with a nucleophile on the macromolecule is a standard practice. Previously, we have used the site-directed incorporation of cysteine nucleophiles at the periphery of an antibody's binding site, paired with the chemical design of weakly electrophilic ligands, to produce receptor-ligand pairs that conjugate specifically and permanently ) Bioconjugate Chem.15, 1392-1402 and references therein). After protein expression in Drosophila S2 cells we found, as is frequently observed, that the engineered cysteine was reversibly blocked by disulfide linkage to a cysteine monomer (cysteinylated). Removal of the cysteine monomer requires some care because of the need to preserve other disulfide linkages in the protein. Here we report that cysteinylation can be used to advantage by treating the cysteine monomer as a leaving group and the protein disulfide as an electrophile with special affinity for thiols. Two ligands bearing thiol side chains were synthesized and incubated with the cysteinylated antibody Fab fragment 2D12.5 G54C, with the finding that both ligands become covalently attached within a few minutes under physiological conditions. The attachment is robust even in the presence of excess thiol reagents. This rapid, specific conjugation is particularly interesting for biomedical applications.

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Site-directed ligand discovery

Cited by 445 publications

References 32 publications

Dissecting allosteric effects of activator–coactivator complexes using a covalent small molecule ligand

Dissecting allosteric effects of activator–coactivator complexes using a covalent small molecule ligand

Small-Molecule Inhibitors of IL-2/IL-2R: Lessons Learned and Applied

Cysteinylated Protein as Reactive Disulfide: An Alternative Route to Affinity Labeling

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