Donghui Zhou

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

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 VTNSMB·uPAR 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.

Probing binding and cellular activity of pyrrolidinone and piperidinone small molecules targeting the urokinase receptor.

The urokinase receptor (uPAR) is a cell‐surface protein that is part of an intricate web of transient and tight protein interactions that promote cancer cell invasion and metastasis. Here, we evaluate the binding and biological activity of a new class of pyrrolidinone and piperidinone compounds, along with derivatives of previously‐identified pyrazole and propylamine compounds. Competition assays revealed that the compounds displace a fluorescently labeled peptide (AE147‐FAM) with inhibition constant (Ki) values ranging from 6 to 63 μM. Structure‐based computational pharmacophore analysis followed by extensive explicit‐solvent molecular dynamics (MD) simulations and free energy calculations suggested the pyrazole‐based and piperidinone‐based compounds adopt different binding modes, despite their similar two‐dimensional structures. In cells, pyrazole‐based compounds showed significant inhibition of breast adenocarcinoma (MDA‐MB‐231) and pancreatic ductal adenocarcinoma (PDAC) cell proliferation, but piperidinone‐containing compounds exhibited no cytotoxicity even at concentrations of 100 μM. One pyrazole‐based compound impaired MDA‐MB‐231 invasion, adhesion, and migration in a concentration‐dependent manner, while the piperidinone inhibited only invasion. The pyrazole derivative inhibited matrix metalloprotease‐9 (gelatinase) activity in a concentration‐dependent manner, while the piperidinone showed no effect suggesting different mechanisms for inhibition of cell invasion. Signaling studies further highlighted these differences, showing that pyrazole compounds completely inhibited ERK phosphorylation and impaired HIF1α and NF‐κB signaling, while pyrrolidinones and piperidinones had no effect. Annexin V staining suggested that the effect of the pyrazole‐based compound on proliferation was due to cell killing through an apoptotic mechanism. The compounds identified represent valuable leads in the design of further derivatives with higher affinities and potential probes to unravel the protein–protein interactions of uPAR.

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

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.

Structure-Based Target-Specific Screening Leads to Small-Molecule CaMKII Inhibitors

Target‐specific scoring methods are more commonly used to identify small‐molecule inhibitors among compounds docked to a target of interest. Top candidates that emerge from these methods have rarely been tested for activity and specificity across a family of proteins. In this study we docked a chemical library into CaMKIIδ, a member of the Ca2+/calmodulin (CaM)‐dependent protein kinase (CaMK) family, and re‐scored the resulting protein–compound structures using Support Vector Machine SPecific (SVMSP), a target‐specific method that we developed previously. Among the 35 selected candidates, three hits were identified, such as quinazoline compound 1 (KIN‐1; N4‐[7‐chloro‐2‐[(E)‐styryl]quinazolin‐4‐yl]‐N1,N1‐diethylpentane‐1,4‐diamine), which was found to inhibit CaMKIIδ kinase activity at single‐digit micromolar IC50. Activity across the kinome was assessed by profiling analogues of 1, namely 6 (KIN‐236; N4‐[7‐chloro‐2‐[(E)‐2‐(2‐chloro‐4,5‐dimethoxyphenyl)vinyl]quinazolin‐4‐yl]‐N1,N1‐diethylpentane‐1,4‐diamine), and an analogue of hit compound 2 (KIN‐15; 2‐[4‐[(E)‐[(5‐bromobenzofuran‐2‐carbonyl)hydrazono]methyl]‐2‐chloro‐6‐methoxyphenoxy]acetic acid), namely 14 (KIN‐332; N‐[(E)‐[4‐(2‐anilino‐2‐oxoethoxy)‐3‐chlorophenyl]methyleneamino]benzofuran‐2‐carboxamide), against 337 kinases. Interestingly, for compound 6, CaMKIIδ and homologue CaMKIIγ were among the top ten targets. Among the top 25 targets of 6, IC50 values ranged from 5 to 22 μm. Compound 14 was found to be not specific toward CaMKII kinases, but it does inhibit two kinases with sub‐micromolar IC50 values among the top 25. Derivatives of 1 were tested against several kinases including several members of the CaMK family. These data afforded a limited structure–activity relationship study. Molecular dynamics simulations with explicit solvent followed by end‐point MM‐GBSA free‐energy calculations revealed strong engagement of specific residues within the ATP binding pocket, and also changes in the dynamics as a result of binding. This work suggests that target‐specific scoring approaches such as SVMSP may hold promise for the identification of small‐molecule kinase inhibitors that exhibit some level of specificity toward the target of interest across a large number of proteins.

Small-molecule CaVα1⋅CaVβ antagonist suppresses neuronal voltage-gated calcium-channel trafficking

Significance Voltage-gated ion channels, such as CaV2.2, consist of pore-forming and auxiliary subunits that interact through protein–protein interactions. We develop a small-molecule antagonist of the protein–protein interaction between the calcium channel alpha pore-forming domain (CaVα) and beta subunits (CaVβ). The compound suppresses trafficking of CaV2.2 channels to the cell membrane and inhibits CaV2.2 activity by acting intracellularly. This allows peripheral access and eliminates the need of intrathecal administration. Indeed, in vivo systemic administration of the small molecule reduces neuropathic pain behavior in animal models. Our compounds serve as chemical tools to explore the CaVα⋅CaVβ interaction in vivo and as a starting point for the development of therapeutics for the treatment of a range of disorders associated with calcium channels. Extracellular calcium flow through neuronal voltage-gated CaV2.2 calcium channels converts action potential-encoded information to the release of pronociceptive neurotransmitters in the dorsal horn of the spinal cord, culminating in excitation of the postsynaptic central nociceptive neurons. The CaV2.2 channel is composed of a pore-forming α1 subunit (CaVα1) that is engaged in protein–protein interactions with auxiliary α2/δ and β subunits. The high-affinity CaV2.2α1⋅CaVβ3 protein–protein interaction is essential for proper trafficking of CaV2.2 channels to the plasma membrane. Here, structure-based computational screening led to small molecules that disrupt the CaV2.2α1⋅CaVβ3 protein–protein interaction. The binding mode of these compounds reveals that three substituents closely mimic the side chains of hot-spot residues located on the α-helix of CaV2.2α1. Site-directed mutagenesis confirmed the critical nature of a salt-bridge interaction between the compounds and CaVβ3 Arg-307. In cells, compounds decreased trafficking of CaV2.2 channels to the plasma membrane and modulated the functions of the channel. In a rodent neuropathic pain model, the compounds suppressed pain responses. Small-molecule α-helical mimetics targeting ion channel protein–protein interactions may represent a strategy for developing nonopioid analgesia and for treatment of other neurological disorders associated with calcium-channel trafficking.