Probing Binding and Cellular Activity of Pyrrolidinone and Piperidinone Small Molecules Targeting the Urokinase Receptor

Mani, Timmy; Liu, Degang; Zhou, Dongfang; Li, Liwei; Knabe, William E.; Wang, Fang; Oh, Kyungsoo; Meroueh, Samy O.

doi:10.1002/cmdc.201300340

Cited by 21 publications

(27 citation statements)

References 57 publications

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“…Duplicates were removed using ZINC codes, leading to a set of 716113 with unique ZINC codes. A substructure search was carried out on the core structure of pyrazole, piperidinone, and pyrrolidinone compounds that were previously shown to bind to uPAR with inhibition constants ∼ 25 µM (45,50). The fragments that were used for the search are shown in Scheme S1.…”

Section: Methodsmentioning

confidence: 99%

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A New Class of Orthosteric uPAR·uPA Small-Molecule Antagonists Are Allosteric Inhibitors of the uPAR·Vitronectin Interaction

et al. 2015

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The urokinase receptor (uPAR) is a GPI-anchored cell surface receptor that is at the center of an intricate network of protein-protein interactions. Its immediate binding partners are the serine proteinase urokinase (uPA), and vitronectin (VTN), a component of the extracellular matrix. uPA and VTN bind at distinct sites on uPAR to promote extracellular matrix degradation and integrin signaling, respectively. Here, we report the discovery of a new class of pyrrolone small-molecule inhibitors of the tight ∼1 nM uPAR•uPA protein-protein interaction. These compounds were designed to bind to the uPA pocket on uPAR. The highest affinity compound, namely 7, displaced a fluorescently-labeled α-helical peptide (AE147-FAM) with an inhibition constant Ki of 0.7 µM and inhibited the tight uPAR•uPAATF interaction with an IC50 of 18 µM. Biophysical studies with surface plasmon resonance showed that VTN binding is highly dependent on uPA. This cooperative binding was confirmed as 7, which binds at the uPAR•uPA interface, also inhibited the distal VTN•uPAR interaction. In cell culture, 7 blocked the uPAR•uPA interaction in uPAR-expressing human embryonic kidney (HEK-293) cells, and impaired cell adhesion to VTN, a process that is mediated by integrins. As a result, 7 inhibited integrin signaling in MDA-MB-231 cancer cells as evidenced by a decrease in focal adhesion kinase (FAK) phosphorylation and Rac1 GTPase activation. Consistent with these results, 7 blocked breast MDA-MB-231 cancer cell invasion with IC50 values similar to those observed in ELISA and surface plasmon resonance competition studies. Explicit-solvent molecular dynamics simulations show that the cooperativity between uPA and VTN is attributed to stabilization of uPAR motion by uPA. In addition, free energy calculations revealed that uPA stabilizes the VTN•uPARSMB interaction through more favorable electrostatics and entropy. Disruption of the uPAR•VTNSMB interaction by 7 is consistent with the cooperative binding to uPAR by uPA and VTN. Interestingly, the VTNSMB•uPAR interaction was less favorable in the VTNSMB•uPAR•7 complex suggesting potential cooperativity between 7 and VTN. Compound 7 provides an excellent starting point for the development of more potent derivatives to explore uPAR biology.

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Section: Methodsmentioning

confidence: 99%

“…IPR-803 inhibited cancer cell invasion and blocked cancer metastasis in vivo (43). Our work has shown that compounds that bind to uPAR share common structural features and occupy specific pockets in uPAR that accommodate critical hot-spot residues of uPA (45). …”

Section: Introductionmentioning

confidence: 99%

A New Class of Orthosteric uPAR·uPA Small-Molecule Antagonists Are Allosteric Inhibitors of the uPAR·Vitronectin Interaction

et al. 2015

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“…23 Interestingly, these compounds were identified in a ligand-based approach using the structure of pyrazole, pyrrolodinone, piperidinone, and butan-amine compounds that were identified by structure-based virtual screening using the structure of uPAR from the uPAR· uPA complex. 21 The carbon that bears the hydroxyl group of the pyrrolinone compounds considered in this work may be prone to attack by nucleophiles. However, the OH group makes a poor leaving group, making covalent adduct formation between the compound and protein unlikely.…”

Section: Discussionmentioning

confidence: 99%

“…18,21,22 Compound 1 was discovered following a substructure search using pyrazole, piperidinone, and pyrrolydi-none compounds as templates. 23 These compounds were shown to bind to uPAR and displace a fluorescently labeled peptide.…”

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confidence: 99%

Small Molecules Engage Hot Spots through Cooperative Binding To Inhibit a Tight Protein–Protein Interaction

Liu

et al. 2017

Biochemistry

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Protein–protein interactions drive every aspect of cell signaling, yet only a few small-molecule inhibitors of these interactions exist. Despite our ability to identify critical residues known as hot spots, little is known about how to effectively engage them to disrupt protein–protein interactions. Here, we take advantage of the ease of preparation and stability of pyrrolinone 1, a small-molecule inhibitor of the tight interaction between the urokinase receptor (uPAR) and its binding partner, the urokinase-type plasminogen activator uPA, to synthesize more than 40 derivatives and explore their effect on the protein–protein interaction. We report the crystal structure of uPAR bound to previously discovered pyrazole 3 and to pyrrolinone 12. While both 3 and 12 bind to uPAR and compete with a fluorescently labeled peptide probe, only 12 and its derivatives inhibit the full uPAR·uPA interaction. Compounds 3 and 12 mimic and engage different hot-spot residues on uPA and uPAR, respectively. Interestingly, 12 is involved in a π–cation interaction with Arg-53, which is not considered a hot spot. Explicit-solvent molecular dynamics simulations reveal that 3 and 12 exhibit dramatically different correlations of motion with residues on uPAR. Free energy calculations for the wild-type and mutant uPAR bound to uPA or 12 show that Arg-53 interacts with uPA or with 12 in a highly cooperative manner, thereby altering the contributions of hot spots to uPAR binding. The direct engagement of peripheral residues not considered hot spots through π–cation or salt-bridge interactions could provide new opportunities for enhanced small-molecule engagement of hot spots to disrupt challenging protein–protein interactions.

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“…Pyrazole-based compounds that bind to and inhibit uPAR inhibit the proliferation, invasion, and migration of MDA-MB-231 breast cancer cells via an increase in apoptosis [10]. Similarly, peptides that interfere with the minimal sequence of uPAR required to induce cell motility and angiogenic responses also demonstrated efficacy in vitro in HUVEC and fibrosarcoma cell lines [11].…”

Section: Inhibition Of the Plasminogen Activator System As Cancer Thementioning

confidence: 99%

Emerging pathophysiological roles for fibrinolysis

Rein-Smith

Church²

2014

Current Opinion in Hematology

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Current evidence suggests an association between the plasminogen activator system and its inhibitors in a variety of malignant and inflammatory states. Newly discovered roles for plasminogen activators and plasminogen activator inhibitors in these diseases provide novel targets for future therapeutic development. Additionally, the newly characterized regulation of the plasminogen activator system by endogenous microRNAs provides new insight into the physiological role of this system and its role in disease.

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Probing Binding and Cellular Activity of Pyrrolidinone and Piperidinone Small Molecules Targeting the Urokinase Receptor

Cited by 21 publications

References 57 publications

A New Class of Orthosteric uPAR·uPA Small-Molecule Antagonists Are Allosteric Inhibitors of the uPAR·Vitronectin Interaction

A New Class of Orthosteric uPAR·uPA Small-Molecule Antagonists Are Allosteric Inhibitors of the uPAR·Vitronectin Interaction

Small Molecules Engage Hot Spots through Cooperative Binding To Inhibit a Tight Protein–Protein Interaction

Emerging pathophysiological roles for fibrinolysis

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