Halogen-Enriched Fragment Libraries as Leads for Drug Rescue of Mutant p53

Wilcken, Rainer; Liu, Xiangrui; Zimmermann, Markus O.; Rutherford, Trevor J.; Fersht, Alan R.; Joerger, A.C.; Boeckler, Frank M.

doi:10.1021/ja301056a

Cited by 208 publications

(294 citation statements)

References 33 publications

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“…We analyzed the inhibition of aggregation by examples from two classes of drug leads: PK083 (8), a carbazole derivative, and four new leads, PK5174, PK5176, PK5196, and PK5201 (abbreviated to 83, 5174 etc. ), which are designed to occupy a more extended binding site (19) (Fig. S5).…”

Section: Resultsmentioning

confidence: 99%

“…These values were very similar to those measured directly by isothermal titration calorimetry (ITC). (19) The same was done for k 2 using values of (0.319 AE 0.001 min and for the value for ligand-bound protein (0.055 AE 0.015 min −1 , Fig. 5), to generate values of K I that were, generally, significantly higher, apart from 5176, where the data were poorer.…”

Section: Resultsmentioning

confidence: 99%

“…But ITC shows a stoichiometry of 1∶1 ligand bound to protein. (19) The remaining alternative is that ligand-bound protein can still aggregate, albeit at a much reduced rate.…”

Section: Discussion Complex Kinetics Fits To a Simple Equation Other mentioning

confidence: 99%

“…(18) In order to design and assay novel, mutant-specific anticancer drugs that can inhibit the aggregation of Y220C, we have analyzed the kinetics of aggregation of the mutant and found it fits a very simple scheme. We have used novel binding ligands (19) as tools to probe the biophysics of denaturation and aggregation in vitro and the structural requirements for inhibition of aggregation. We have found candidates for molecules that can reactivate Y220C in a cancer cell line.…”

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Kinetic mechanism of p53 oncogenic mutant aggregation and its inhibition

Wilcken

Wang

Boeckler

et al. 2012

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

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112

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Aggregation of destabilized mutants of the tumor suppressor p53 is a major route for its loss of activity. In order to assay drugs that inhibit aggregation of p53, we established the basic kinetics of aggregation of its core domain, using the mutant Y220C that has a mutation-induced, druggable cavity. Aggregation monitored by light scattering followed lag kinetics. Electron microscopy revealed the formation of small aggregates that subsequently grew to larger amorphous aggregates. The kinetics of aggregation produced surprising results: progress curves followed either by the binding of Thioflavin T or the fluorescence of the protein at 340 nm fitted well to simple two-step sequential first-order lag kinetics with rate constants k 1 and k 2 that were independent of protein concentration, and not to classical nucleation-growth. We suggest a mechanism of first-order formation of an aggregation competent state as being rate determining followed by rapid polymerization with the higher order kinetics. By measuring the inhibition kinetics of k 1 and k 2 , we resolved that the process with the higher rate constant followed that of the lower. Further, there was only partial inhibition of k 1 and k 2 , which showed two parallel pathways of aggregation, one via a state that requires unfolding of the protein and the other of partial unfolding with the ligand still bound. Inhibition kinetics of ligands provides a useful tool for probing an aggregation mechanism.amyloid | misfolding | folding | cancer T he most frequently mutated gene in cancer is that of the tumor suppressor p53. Some 70% of types of human cancers have their p53 directly inactivated by mutation, and so its reactivation is an important therapeutic goal (1). The most common oncogenic mutations are of residues in the DNA binding domain (DBD, residues 94-312) that make direct contact with DNA (2). But about 30%-40% of oncogenic mutants are inactivated because the mutations lower the stability of the DBD so that it denatures at body temperature, although it can be fully active at lower temperatures (3-6). We are attempting to rescue those temperature-sensitive mutants of p53 by designing small molecules that bind specifically to the native state of the protein and stabilize it (7). The rationale is that small molecules that bind to the native state of a temperature-sensitive mutant and not the denatured states will raise the melting temperature by a mass action effect. Such a molecule will slow down the rate of unfolding if it binds weakly to the transition state for unfolding. The obvious binding sites to target are those already present that bind natural ligands. In theory, they can be targeted in a chaperone strategy in which a rescue drug will compete for a binding site (7). We have chosen, instead, as a particularly useful paradigm for these studies, the stability of the p53 mutant Y220C, because the mutation induces a druggable cavity that is remote from functional areas of the protein (8, 9). We have designed small molecules that raise the apparent T m of ...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

“…But ITC shows a stoichiometry of 1∶1 ligand bound to protein. (19) The remaining alternative is that ligand-bound protein can still aggregate, albeit at a much reduced rate.…”

Section: Discussion Complex Kinetics Fits To a Simple Equation Other mentioning

confidence: 99%

mentioning

confidence: 99%

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Kinetic mechanism of p53 oncogenic mutant aggregation and its inhibition

Wilcken

Wang

Boeckler

et al. 2012

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

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112

137

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“…We initially tackled the rescue by stabilizing the folded conformation against unfolding, first by a generic approach using a peptide that binds to native p53 (34) and mutants (35) and then by targeting a specific mutant where the mutation forms a druggable cavity, Y220C. Its melting temperature is raised, and its aggregation is slowed in vitro by small mutant-specific molecules that bind in the cavity (36, 37) and rescue Y220C in cancer cell lines (25,38,39). Eisenberg and coworkers (40) have taken a different approach of inhibiting the aggregation process, per se, with a peptide that caps the specific exposed aggregation-prone sequence, centered on Ile254, rather than stabilizing the native structure.…”

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Multisite aggregation of p53 and implications for drug rescue

Wang

Fersht

2017

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

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Protein aggregation is involved in many diseases. Often, a unique aggregation-prone sequence polymerizes to form regular fibrils. Many oncogenic mutants of the tumor suppressor p53 rapidly aggregate but form amorphous fibrils. A peptide surrounding Ile254 is proposed to be the aggregation-driving sequence in cells. We identified several different aggregating sites from limited proteolysis of harvested aggregates and effects of mutations on kinetics and products of aggregation. We present a model whereby the amorphous nature of the aggregates results from multisite branching of polymerization after slow unfolding of the protein, which may be a common feature of aggregation of large proteins. Greatly lowering the aggregation propensity of any one single site, including the site of Ile254, by mutation did not inhibit aggregation in vitro because aggregation could still occur via the other sites. Inhibition of an individual site is, accordingly, potentially unable to prevent aggregation in vivo. However, cancer cells are specifically killed by peptides designed to inhibit the Ile254 sequence and further aggregation-driving sequences that we have found. Consistent with our proposed mechanism of aggregation, we found that such peptides did not inhibit aggregation of mutant p53 in vitro. The cytotoxicity was not eliminated by knockdown of p53 in 2D cancer cell cultures. The peptides caused rapid cell death, much faster than usually expected for p53-mediated transcription-dependent apoptosis. There may also be non-p53 targets for those peptides in cancer cells, such as p63, or the peptides may alter other interactions of partly denatured p53 with receptors.amyloid | mechanism | misfolding | disease T he tumor suppressor p53 is inactivated by mutation in a substantial number of tumors (1-3). Some 30-40% of those oncogenic mutants are simply destabilized by mutations in its core domain. Those mutants are temperature-sensitive, having a WT structure at lower temperatures, but melt at close to body temperature or below and rapidly aggregate (4-6). Protein aggregation occurs in many diseases (7-9). The best mechanistically characterized examples involve the polymerization of aggregationprone peptides (10, 11) or small proteins to give well-defined fibrils based on a regular repeat structure (12-18). The fibrillar aggregates have a characteristic cross-β X-ray diffraction pattern and bind such dyes as Congo Red and Thioflavine T (ThT) (16). The kinetics of aggregation usually follow a nucleation-growth mechanism, with very slow nucleation (19-21). WT p53 itself aggregates at body temperature (4-6, 22-24), and the oncogenic destabilized mutants aggregate even faster to give amorphous structures that display the characteristic diffraction pattern and bind those diagnostic dyes (23, 25), although under certain conditions, such as very high pressure, they will generate regular fibrils (23,26). The mechanism of initiation of aggregation of p53 differs from the usually studied examples. Two molecules of the core domain of p53 exten...

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Molecular Interaction and Recognition

Daze

Hof

2016

Encyclopedia of Physical Organic Chemistry, 5 Volume Set

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Halogen-Enriched Fragment Libraries as Leads for Drug Rescue of Mutant p53

Cited by 208 publications

References 33 publications

Kinetic mechanism of p53 oncogenic mutant aggregation and its inhibition

Kinetic mechanism of p53 oncogenic mutant aggregation and its inhibition

Multisite aggregation of p53 and implications for drug rescue

Molecular Interaction and Recognition

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