Design and Applications of Bifunctional Small Molecules: Why Two Heads Are Better Than One

Corson, Timothy W.; Aberle, Nicholas S.; Crews, Craig M.

doi:10.1021/cb8001792

Cited by 139 publications

(133 citation statements)

References 96 publications

(136 reference statements)

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“…Because both the C2-C3 and C7-C8 olefins are necessary to observe the levels of toxicity seen for PL, we speculated that multivalency-the ability to interact with multiple cellular targets or a single cellular target at more than one location-might alter toxicity in this system (13)(14)(15)(16). Thus, we synthesized a structurally analogous PL"monomer," "dimer," and "trimer" using a Mitsunobu approach (Fig.…”

Section: Resultsmentioning

confidence: 99%

Synthesis, cellular evaluation, and mechanism of action of piperlongumine analogs

Adams

Dai

Pellegrino

et al. 2012

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

208

221

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Piperlongumine is a naturally occurring small molecule recently identified to be toxic selectively to cancer cells in vitro and in vivo. This compound was found to elevate cellular levels of reactive oxygen species (ROS) selectively in cancer cell lines. The synthesis of 80 piperlongumine analogs has revealed structural modifications that retain, enhance, and ablate key piperlongumine-associated effects on cells, including elevation of ROS, cancer cell death, and selectivity for cancer cells over nontransformed cell types. Structure/activity relationships suggest that the electrophilicity of the C2-C3 olefin is critical for the observed effects on cells. Furthermore, we show that analogs lacking a reactive C7-C8 olefin can elevate ROS to levels observed with piperlongumine but show markedly reduced cell death, suggesting that ROS-independent mechanisms, including cellular cross-linking events, may also contribute to piperlongumine's induction of apoptosis. In particular, we have identified irreversible protein glutathionylation as a process associated with cellular toxicity. We propose a mechanism of action for piperlongumine that may be relevant to other small molecules having two sites of reactivity, one with greater and the other with lesser electrophilicity.

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Section: Resultsmentioning

confidence: 99%

Synthesis, cellular evaluation, and mechanism of action of piperlongumine analogs

Adams

Dai

Pellegrino

et al. 2012

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

208

221

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“…Combining the attractive qualities of both RNAi and small molecule inhibitor approaches, we developed a strategy, proteolysis targeting chimeras (PROTACs) for targeted posttranslational knockdown of proteins. Each PROTAC is heterodimeric, consisting of an E3 ubiquitin ligase-binding moiety linked to a ligand that binds to the target protein (3). As such, each PROTAC recruits its target protein to the E3 ubiquitin ligase, resulting in target protein ubiquitination by the E3 ligase and, ultimately, proteasome-mediated degradation of the target (4-6).…”

mentioning

confidence: 99%

Posttranslational protein knockdown coupled to receptor tyrosine kinase activation with phosphoPROTACs

Hines

Gough

Corson

et al. 2013

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

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Posttranslational knockdown of a specific protein is an attractive approach for examining its function within a system. Here we introduce phospho-dependent proteolysis targeting chimeras (phosphoPROTACs), a method to couple the conditional degradation of targeted proteins to the activation state of particular kinasesignaling pathways. We generated two phosphoPROTACs that couple the tyrosine phosphorylation sequences of either the nerve growth factor receptor, TrkA (tropomyosin receptor kinase A), or the neuregulin receptor, ErbB3 (erythroblastosis oncogene B3), with a peptide ligand for the E3 ubiquitin ligase von Hippel Lindau protein. These phosphoPROTACs recruit either the neurotrophic signaling effector fibroblast growth factor receptor substrate 2α or the survival-promoting phosphatidylinositol-3-kinase, respectively, to be ubiquitinated and degraded upon activation of specific receptor tyrosine kinases and phosphorylation of the phosphoPROTACs. We demonstrate the ability of these phosphoPROTACs to suppress the short-and long-term effects of their respective activating receptor tyrosine kinase pathways both in vitro and in vivo. In addition, we show that activation of phosphoPROTACs is entirely dependent on their kinase-mediated phosphorylation, as phenylalanine-containing null variants are inactive. Furthermore, stimulation of unrelated growth factor receptors does not induce target protein knockdown. Although comparable in efficiency to RNAi, this approach has the added advantage of providing a degree of temporal and dosing control as well as cell-type selectivity unavailable using nucleic acid-based strategies. By varying the autophosphorylation sequence of a phosphoPROTAC, it is conceivable that other receptor tyrosine kinase/effector pairings could be similarly exploited to achieve other biological effects.A ntagonizing the function of a protein is an effective strategy for determining its role within a cellular context. For some enzymes and/or receptor proteins, this can be accomplished by incubation with a small molecule inhibitor or antagonist. However, specific small molecule inhibitors have not been discovered or developed for many proteins. As alternative strategies, gene deletion and RNAi approaches function at the DNA and mRNA levels to reduce protein expression, thus offering the ability to control function even for those proteins lacking a specific inhibitor (1, 2). However, such nucleic acid-based approaches do not afford the same rapid, direct assessment of protein function as posttranslational intervention; furthermore, in vivo application of these strategies is even today still complicated and relatively cumbersome. Combining the attractive qualities of both RNAi and small molecule inhibitor approaches, we developed a strategy, proteolysis targeting chimeras (PROTACs) for targeted posttranslational knockdown of proteins. Each PROTAC is heterodimeric, consisting of an E3 ubiquitin ligase-binding moiety linked to a ligand that binds to the target protein (3). As such, each PROTAC recruits i...

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“…This concept is well known in nature as a way to increase affinity and selectivity, such as in virus-cell and antibody-antigen recognition (1). Consequently, linking two ligands together can be a strategy to enhance binding of drug candidates to therapeutically relevant proteins by exploiting a bivalent binding site (2)(3)(4)(5). It is proposed that such dimeric inhibitors will foster a more potent response by increasing the affinity toward their targets by some hundred-fold (6,7).…”

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confidence: 99%

“…PDZ domains constitute a class of protein-protein interacting modules that functions as scaffolds and adapters in signaling cascades, and they are found in a few hundred proteins in the human genome (13). PDZ domains generally bind to the C termini of their target proteins (14,15), although neuronal nitric oxide synthase binds to postsynaptic density protein-95 (PSD-95) 3 via an internally located sequence (14). PDZ domains often occur as concatenates of two or more domains.…”

mentioning

confidence: 99%

Deciphering the Kinetic Binding Mechanism of Dimeric Ligands Using a Potent Plasma-stable Dimeric Inhibitor of Postsynaptic Density Protein-95 as an Example

Celestine

Bach

Gottschalk

et al. 2010

Journal of Biological Chemistry

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Dimeric ligands can be potent inhibitors of protein-protein or enzyme-substrate interactions. They have increased affinity and specificity toward their targets due to their ability to bind two binding sites simultaneously and are therefore attractive in drug design. However, few studies have addressed the kinetic mechanism of interaction of such bivalent ligands. We have investigated the binding interaction of a recently identified potent plasma-stable dimeric pentapeptide and PDZ1-2 of postsynaptic density protein-95 (PSD-95) using protein engineering in combination with fluorescence polarization, isothermal titration calorimetry, and stopped-flow fluorimetry. We demonstrate that binding occurs via a two-step process, where an initial binding to either one of the two PDZ domains is followed by an intramolecular step, which produces the bidentate complex. We have determined all rate constants involved in the binding reaction and found evidence for a conformational transition of the complex. Our data demonstrate the importance of a slow dissociation for a successful dimeric ligand but also highlight the possibility of optimizing the intramolecular association rate. The results may therefore aid the design of dimeric inhibitors in general.Biological molecules that comprise two or more binding units enable di-or multivalent binding to their protein partners. This concept is well known in nature as a way to increase affinity and selectivity, such as in virus-cell and antibody-antigen recognition (1). Consequently, linking two ligands together can be a strategy to enhance binding of drug candidates to therapeutically relevant proteins by exploiting a bivalent binding site (2-5). It is proposed that such dimeric inhibitors will foster a more potent response by increasing the affinity toward their targets by some hundred-fold (6, 7). Indeed, in vitro binding studies have shown improved affinities of dimeric inhibitors toward their targets as compared with their monomeric counterparts (5, 8 -11). It is complex to predict the overall affinity enhancement by linking two ligands because the observed binding energy is not a direct summation of the binding energies of individual components, and the entropy and enthalpy compensation are difficult to estimate (6,7,12). Therefore, experimental determination of the binding mechanism of dimeric ligands is useful for future design of dimeric ligands. However, there are only a few cases where in solution methods have been used to determine the mechanism of interaction of such ligands (5,7,10). One class of proteins where dimeric ligands have been exploited in an attempt to develop potential inhibitors for therapeutically relevant interactions in the cell is the PDZ (PSD-95/Dlg/Zonula occludens-1) domain family of proteins (5, 8). PDZ domains constitute a class of protein-protein interacting modules that functions as scaffolds and adapters in signaling cascades, and they are found in a few hundred proteins in the human genome (13). PDZ domains generally bind to the C termini of thei...

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Design and Applications of Bifunctional Small Molecules: Why Two Heads Are Better Than One

Cited by 139 publications

References 96 publications

Synthesis, cellular evaluation, and mechanism of action of piperlongumine analogs

Synthesis, cellular evaluation, and mechanism of action of piperlongumine analogs

Posttranslational protein knockdown coupled to receptor tyrosine kinase activation with phosphoPROTACs

Deciphering the Kinetic Binding Mechanism of Dimeric Ligands Using a Potent Plasma-stable Dimeric Inhibitor of Postsynaptic Density Protein-95 as an Example

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