Small‐Molecule Inhibition of the uPAR ⋅ uPA Interaction by Conformational Selection

Abstract

The urokinase receptor (uPAR) is a cell surface receptor that binds to the serine protease urokinase‐type plasminogen activator (uPA) with high affinity. This interaction is beneficial for extravascular fibrin clearance, but it has also been associated with a broad range of pathological conditions including cancer, atherosclerosis, and kidney disease. Here, starting with a small molecule that we previously discovered by virtual screening and cheminformatics analysis, we design and synthesize several derivatives that were tested for binding and inhibition of the uPAR ⋅ uPA interaction. To confirm the binding site and establish a binding mode of the compounds, we carried out biophysical studies using uPAR mutants, among them uPARH47C−N259C, a mutant previously developed to mimic the structure of uPA‐bound uPAR. Remarkably, a substantial increase in potency is observed for inhibition of uPARH47C−N259C binding to uPA compared to wild‐type uPAR, consistent with our use of the structure of uPAR in its uPA‐bound state to design small‐molecule uPAR ⋅ uPA antagonists. Combined with the biophysical studies, molecular docking followed by extensive explicit‐solvent molecular dynamics simulations and MM‐GBSA free energy calculations yielded the most favorable binding pose of the compound. Collectively, these results suggest that potent inhibition of uPAR binding to uPA with small molecules will likely only be achieved by developing small molecules that exhibit high‐affinity to solution apo structures of uPAR, rather than uPA‐bound structures of the receptor.

Urokinase-type plasminogen activator receptor (uPAR) as a therapeutic target in cancer

“…Human and mouse uPA1-48 (huPA1-48 and muPA1-48), human and murine uPA1-48 fusion proteins (huPA1-48Ig and muPA1-48Ig) [ 120 ], and human and mouse pegylated uPA1-48 (PEGh1-48 and PEGhm1-48) [ 121 ] also inhibit tumour growth by inhibiting tumour stromal cell uPAR-dependent plasminogen activation. The small-molecule inhibitors IPR-456 [ 122 ], IPR-803 [ 123 ], IPR-3011 [ 124 ], IPR-3577 [ 125 ], 7 [ 126 ], LLL-1fsi [ 127 ], MS#479 [2-(pyridin-2-ylamino)-quinolin-8-ol] and MS#305 [2,2′-(methylimino)di (8-quinolinol)] [ 128 ], Compounds 6 and 37 [ 129 ], and docosahexaenoic acid (DHA) [ 130 ] inhibit the uPAR/uPA, uPAR/integrin, uPAR/Vn or uPAR/FPR interaction. The ligand-targeted toxins DTAT [diphtheria toxin (DT) and ATF] [ 131 , 132 ], DTATEGF (ATF, EGF and DT) [ 133 ], DTAT13 [ATF, interleukin-13 (IL-13) and DT] [ 134 , 135 ], eBAT (EGFATFKDEL 7mut) [ 136 – 141 ], ATF-SAP (ATF and Saporin) [ 142 , 143 ], PAI-2- N -AIE conjugate [5,7-dibromo- N -( p -hydroxymethylbenzyl)isatin and PAI-2] [ 144 ], DTU2GMCSF [DT and granulocyte–macrophage colony-stimulating factor (GM-CSF)] [ 145 ], ATF-PE38 and ATF-PE38KDEL [ATF and Pseudomonas exotoxin A (PE38)] [ 146 ] exert antitumor effects by targeting uPAR and releasing toxins.…”

“…Furthermore, linear peptides based on the sequence of uPA lack potency and have poor pharmacological properties and stability due to susceptibility to exoprotease degradation in the plasma [ 153 ]; screening for small-molecule inhibitors is inefficient due to a lack of detailed structural information on the interactions of uPAR with its binding partners such as integrins [ 154 – 156 ]. Some uPAR-targeted small-molecule inhibitors are hydrophobic and have limited bioavailability [ 123 , 125 , 157 ]; and due to the large surface area at the protein–protein interface, the development of small molecules specifically targeting this flexible hydrophobic cavity in uPAR also represent a challenging task [ 129 , 158 ]. Similarly, ligand-targeted toxins must overcome many barriers before they reach human clinical trials, including determining the appropriate dosing strategy and sequence of administration, increasing the potency and reducing the immunogenicity of the toxin [ 159 , 160 ].…”

Therapeutic Strategies Targeting Urokinase and Its Receptor in Cancer

“…Small molecules inhibiting with high affinity uPAR binding to uPA were constructed. They make uPAR unable to bind uPA, modifying its conformation [145,146]. Using a biophysical approach, molecular docking and extensive explicit-solvent molecular dynamics simulations of uPAR bound to uPA, Xu, D. et al demonstrated that the small compound IPR-3011 blocks uPA binding to both open and closed conformations of uPAR [146].…”

Targeting the Urokinase-Type Plasminogen Activator Receptor (uPAR) in Human Diseases With a View to Non-invasive Imaging and Therapeutic Intervention

“…Building on structural information on the uPA•uPAR interaction, Meroueh et al nevertheless succeeded in developing a small compound (IPR-3011) that inhibited AE147 binding to uPAR with an inhibition constant K i = 2.4 ± 0.3 μM ( Xu et al, 2017 ). Interestingly, that compound inhibited uPA-binding to the open conformation of uPAR wt with a K i = 60 ± 5 μM, while it inhibited uPA-binding to the closed conformation of uPAR H 47 C – N 259 C with a 10-fold improved efficacy K i = 6.6 ± 0.4 μM ( Xu et al, 2021 ).…”