Crystal Packing Reveals a Potential Autoinhibited KRAS Dimer Interface and a Strategy for Small-Molecule Inhibition of RAS Signaling.

KRAS GTPases harbor oncogenic mutations in more than 25% of human tumors. KRAS is considered to be largely undruggable due to the lack of a suitable small-molecule binding site. Here, we report a unique crystal structure of His-tagged KRASG12D that reveals a remarkable conformational change. The Switch I loop of one His-KRASG12D structure extends into the Switch I/II pocket of another His-KRASG12D in an adjacent unit cell to create an elaborate interface that is reminiscent of high-affinity protein-protein complexes. We explore the contributions of amino acids at this interface using alanine-scanning studies with alchemical free energy perturbation calculations based on explicit-solvent molecular dynamics simulations. Several interface amino acids were found to be hot spots as they contributed more than 1.5 kcal/mol to the protein-protein interaction. Computational analysis of the complex revealed the presence of two large binding pockets that possess physicochemical features typically found in pockets considered druggable. Small-molecule binding to these pockets may stabilize this autoinhibited structure of KRAS if it exists in cells to provide a new strategy to inhibit RAS signaling.

Chloroacetamide fragment library screening identifies new scaffolds for covalent inhibition of the TEAD·YAP1 interaction.

Transcriptional enhanced associate domain (TEAD) binding to co-activator yes-associated protein (YAP1) leads to a transcription factor of the Hippo pathway. TEADs are regulated by S-palmitoylation of a conserved cysteine located in a deep well-defined hydrophobic pocket outside the TEAD·YAP1 interaction interface. Previously, we reported the discovery of a small molecule based on the structure of flufenamic acid that binds to the palmitate pocket, forms a covalent bond with the conserved cysteine, and inhibits TEAD4 binding to YAP1. Here, we screen a fragment library of chloroacetamide electrophiles to identify new scaffolds that bind to the palmitate pocket of TEADs and disrupt their interaction with YAP1. Time- and concentration-dependent studies with wild-type and mutant TEAD1-4 provided insight into their reaction rates and binding constants and established the compounds as covalent inhibitors of TEAD binding to YAP1. Binding pose hypotheses were generated by covalent docking revealing that the fragments and compounds engage lower, middle, and upper sub-sites of the palmitate pocket. Our fragments and compounds provide new scaffolds and starting points for the design of derivatives with improved inhibition potency of TEAD palmitoylation and binding to YAP1.

Exploring Covalent Bond Formation at Tyr-82 for Inhibition of Ral GTPase Activation.

Ral RAS GTPases are directly activated by KRAS through a trimeric complex with a guanine exchange factor. Ral is considered undruggable and lacks an accessible cysteine for covalent drug development. Previously we had reported an aryl sulfonyl fluoride fragment that formed a covalent bond at Tyr-82 on Ral and created a deep and well-defined pocket. Here, we explore this pocket further through design and synthesis of several fragment derivatives. The fragment core is modified by introducing tetrahydronaphthalene or benzodioxane rings to enhance affinity and stability of the sulfonyl fluoride reactive group. The deep pocket in the Switch II region is also explored by modifying the aromatic ring of the fragment that is ensconced into the pocket. Compounds 19 (SOF-658) and 26 (SOF-648) formed a single robust adduct specifically at Tyr-82, inhibited Ral GTPase exchange in buffer and in mammalian cells, and blocked invasion of pancreatic ductal adenocarcinoma cancer cells. Compound 19 (SOF-658) was stable in buffer, mouse, and human microsomes suggesting that further optimization could lead to small molecules to probe Ral activity in tumor models.

Covalent Fragment Inhibits RhoA Activation by Guanine Exchange Factors.

Ras homolog gene family member (RhoA) is a GTPase and a member of the RAS superfamily of GTPases. RhoA is a master regulator of the actin cytoskeleton. It inhibits axon growth preventing repair and recovery following spinal cord and traumatic brain injuries. Despite decades of research into the biological function of Rho GTPases, there exist no small-molecule Rho inhibitors. Here, we screen a library of cysteine electrophiles to explore whether covalent bond formation at Cys-107 leads to inhibition of RhoA activation by guanine exchange factor Trio. Two fragments, propiolamide 1 (ACR-895) and acrylamide 2 (ACR-917), inhibited RhoA nucleotide exchange by Trio in a time-dependent manner. The fragments formed a covalent bond with wild-type RhoA but not Cys107Ser RhoA mutant. Time- and concentration-dependent studies led to equilibrium constants KIs and reaction rates that correspond to t1/2 values in the single-digit hour range. One fragment was selective for RhoA over Rac1 GTPase and had no effect on KRAS nucleotide exchange by SOS1. The fragments did not inhibit RhoA binding to ROCK effector protein. This work establishes Cys-107 as a suitable site for Rho GTPase inhibition and provides fragment starting points for the future development of Rho GTPase covalent inhibitors that could have profound implications in the treatment of patients with injuries of the central nervous system.

Small-Molecule Cyanamide Pan-TEAD·YAP1 Covalent Antagonists.

Transcriptional enhanced associate domains (TEADs) are transcription factors that bind to cotranscriptional activators like the yes-associated protein (YAP) or its paralog transcriptional coactivator with a PDZ-binding motif (TAZ). TEAD·YAP/TAZ target genes are involved in tissue and immune homeostasis, organ size control, tumor growth, and metastasis. Here, we report isoindoline and octahydroisoindole small molecules with a cyanamide electrophile that forms a covalent bond with a conserved cysteine in the TEAD palmitate-binding cavity. Time- and concentration-dependent studies against TEAD1-4 yielded second-order rate constants kinact/KI greater than 100 M-1 s-1. Compounds inhibited YAP1 binding to TEADs with submicromolar IC50 values. Cocrystal structures with TEAD2 enabled structure-activity relationship studies. In mammalian cells, compounds suppressed CTGF mRNA levels and inhibited TEAD1-4 transcriptional activity with submicromolar IC50 values. Inhibition of TEAD binding to YAP1 in mammalian cells was also observed. Several compounds inhibited the cell viability of sarcoma, hepatocellular carcinoma, glioblastoma, and breast cancer cells with single-digit micromolar IC50 values.

Covalent Fragment Screening Identifies Rgl2 RalGEF Cysteine for Targeted Covalent Inhibition of Ral GTPase Activation.

Ral GTPases belong to the RAS superfamily, and they are directly activated by K-RAS. The RalGEF pathway is one of the three major K-RAS signaling pathways. Ral GTPases do not possess a cysteine nucleophile to develop a covalent inhibitor following the strategy that led to a K-RAS G12C therapeutic agent. However, several cysteine amino acids exist on the surface of guanine exchange factors that activate Ral GTPases, such as Rgl2. Here, we screen a library of cysteine electrophile fragments to determine if covalent bond formation at one of the Rgl2 surface cysteines could inhibit Ral GTPase activation. We found several chloroacetamide and acrylamide fragments that inhibited Ral GTPase exchange by Rgl2. Site-directed mutagenesis showed that covalent bond formation at Cys-284, but not other cysteines, leads to inhibition of Ral activation by Rgl2. Follow-up time- and concentration-dependent studies of derivatives identified by substructure search of commercial libraries further confirmed Cys-284 as the reaction site and identified the indoline fragments as the most promising series for further development. Cys-284 is located outside of the Ral ⋅ Rgl2 interface on a loop that has several residues that come in direct contact with Ral GTPases. Our allosteric covalent fragment inhibitors provide a starting point for the development of small-molecule covalent inhibitors to probe Ral GTPases in animal models.

Atractylenolide I enhances responsiveness to immune checkpoint blockade therapy by activating tumor antigen presentation.

One of the primary mechanisms of tumor cell immune evasion is the loss of antigenicity, which arises due to lack of immunogenic tumor antigens as well as dysregulation of the antigen processing machinery. In a screen for small-molecule compounds from herbal medicine that potentiate T cell-mediated cytotoxicity, we identified atractylenolide I (ATT-I), which substantially promotes tumor antigen presentation of both human and mouse colorectal cancer (CRC) cells and thereby enhances the cytotoxic response of CD8+ T cells. Cellular thermal shift assay (CETSA) with multiplexed quantitative mass spectrometry identified the proteasome 26S subunit non-ATPase 4 (PSMD4), an essential component of the immunoproteasome complex, as a primary target protein of ATT-I. Binding of ATT-I with PSMD4 augments the antigen-processing activity of immunoproteasome, leading to enhanced MHC-I-mediated antigen presentation on cancer cells. In syngeneic mouse CRC models and human patient-derived CRC organoid models, ATT-I treatment promotes the cytotoxicity of CD8+ T cells and thus profoundly enhances the efficacy of immune checkpoint blockade therapy. Collectively, we show here that targeting the function of immunoproteasome with ATT-I promotes tumor antigen presentation and empowers T cell cytotoxicity, thus elevating the tumor response to immunotherapy.

Small-Molecule Inhibition of the uPAR ⋠uPA Interaction by Conformational Selection.

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.

Phenotypic Screening of Chemical Libraries Enriched by Molecular Docking to Multiple Targets Selected from Glioblastoma Genomic Data.

Like most solid tumors, glioblastoma multiforme (GBM) harbors multiple overexpressed and mutated genes that affect several signaling pathways. Suppressing tumor growth of solid tumors like GBM without toxicity may be achieved by small molecules that selectively modulate a collection of targets across different signaling pathways, also known as selective polypharmacology. Phenotypic screening can be an effective method to uncover such compounds, but the lack of approaches to create focused libraries tailored to tumor targets has limited its impact. Here, we create rational libraries for phenotypic screening by structure-based molecular docking chemical libraries to GBM-specific targets identified using the tumor's RNA sequence and mutation data along with cellular protein-protein interaction data. Screening this enriched library of 47 candidates led to several active compounds, including 1 (IPR-2025), which (i) inhibited cell viability of low-passage patient-derived GBM spheroids with single-digit micromolar IC50 values that are substantially better than standard-of-care temozolomide, (ii) blocked tube-formation of endothelial cells in Matrigel with submicromolar IC50 values, and (iii) had no effect on primary hematopoietic CD34+ progenitor spheroids or astrocyte cell viability. RNA sequencing provided the potential mechanism of action for 1, and mass spectrometry-based thermal proteome profiling confirmed that the compound engages multiple targets. The ability of 1 to inhibit GBM phenotypes without affecting normal cell viability suggests that our screening approach may hold promise for generating lead compounds with selective polypharmacology for the development of treatments of incurable diseases like GBM.

Small-molecule covalent bond formation at tyrosine creates a binding site and inhibits activation of Ral GTPases.

Ral (Ras-like) GTPases are directly activated by oncogenic Ras GTPases. Mutant K-Ras (G12C) has enabled the development of covalent K-Ras inhibitors currently in clinical trials. However, Ral, and the overwhelming majority of mutant oncogenic K-Ras, are devoid of a druggable pocket and lack an accessible cysteine for the development of a covalent inhibitor. Here, we report that covalent bond formation by an aryl sulfonyl fluoride electrophile at a tyrosine residue (Tyr-82) inhibits guanine exchange factor Rgl2-mediated nucleotide exchange of Ral GTPase. A high-resolution 1.18-Å X-ray cocrystal structure shows that the compound binds to a well-defined binding site in RalA as a result of a switch II loop conformational change. The structure, along with additional high-resolution crystal structures of several analogs in complex with RalA, confirm the importance of key hydrogen bond anchors between compound sulfone oxygen atoms and Ral backbone nitrogen atoms. Our discovery of a pocket with features found on known druggable sites and covalent modification of a bystander tyrosine residue present in Ral and Ras GTPases provide a strategy that could lead to therapeutic agent targeting oncogenic Ras mutants that are devoid of a cysteine nucleophile.

Small-Molecule Covalent Modification of Conserved Cysteine Leads to Allosteric Inhibition of the TEAD⋠Yap Protein-Protein Interaction.

The Hippo pathway coordinates extracellular signals onto the control of tissue homeostasis and organ size. Hippo signaling primarily regulates the ability of Yap1 to bind and co-activate TEA domain (TEAD) transcription factors. Yap1 tightly binds to TEAD4 via a large flat interface, making the development of small-molecule orthosteric inhibitors highly challenging. Here, we report small-molecule TEAD⋅Yap inhibitors that rapidly and selectively form a covalent bond with a conserved cysteine located within the unique deep hydrophobic palmitate-binding pocket of TEADs. Inhibition of TEAD4 binding to Yap1 by these compounds was irreversible and occurred on a longer time scale. In mammalian cells, the compounds formed a covalent complex with TEAD4, inhibited its binding to Yap1, blocked its transcriptional activity, and suppressed expression of connective tissue growth factor. The compounds inhibited cell viability of patient-derived glioblastoma spheroids, making them suitable as chemical probes to explore Hippo signaling in cancer.

Small molecules inhibit ex vivo tumor growth in bone.

Bone is a common site of metastasis for breast, prostate, lung, kidney and other cancers. Bone metastases are incurable, and substantially reduce patient quality of life. To date, there exists no small-molecule therapeutic agent that can reduce tumor burden in bone. This is partly attributed to the lack of suitable in vitro assays that are good models of tumor growth in bone. Here, we take advantage of a novel ex vivo model of bone colonization to report a series of pyrrolopyrazolone small molecules that inhibit cancer cell invasion and ex vivo tumor growth in bone at single-digit micromolar concentration. We find that the compounds modulated the expression levels of genes associated with bone-forming osteoblasts, bone-destroying osteoclasts, cancer cell viability and metastasis. Our compounds provide chemical tools to uncover novel targets and pathways associated with bone metastasis, as well as for the development of compounds to prevent and reverse bone tumor growth in vivo.

Chemical Proteomics Reveals Soluble Epoxide Hydrolase as a Therapeutic Target for Ocular Neovascularization.

The standard-of-care therapeutics for the treatment of ocular neovascular diseases like wet age-related macular degeneration (AMD) are biologics targeting vascular endothelial growth factor signaling. There are currently no FDA approved small molecules for treating these blinding eye diseases. Therefore, therapeutic agents with novel mechanisms are critical to complement or combine with existing approaches. Here, we identified soluble epoxide hydrolase (sEH), a key enzyme for epoxy fatty acid metabolism, as a target of an antiangiogenic homoisoflavonoid, SH-11037. SH-11037 inhibits sEH in vitro and in vivo and docks to the substrate binding cleft in the sEH hydrolase domain. sEH levels and activity are up-regulated in the eyes of a choroidal neovascularization (CNV) mouse model. sEH is overexpressed in human wet AMD eyes, suggesting that sEH is relevant to neovascularization. Known sEH inhibitors delivered intraocularly suppressed CNV. Thus, by dissecting a bioactive compound's mechanism, we identified a new chemotype for sEH inhibition and characterized sEH as a target for blocking the CNV that underlies wet AMD.

Small molecules inhibit STAT3 activation, autophagy, and cancer cell anchorage-independent growth.

Triple-negative breast cancers (TNBCs) lack the signature targets of other breast tumors, such as HER2, estrogen receptor, and progesterone receptor. These aggressive basal-like tumors are driven by a complex array of signaling pathways that are activated by multiple driver mutations. Here we report the discovery of 6 (KIN-281), a small molecule that inhibits multiple kinases including maternal leucine zipper kinase (MELK) and the non-receptor tyrosine kinase bone marrow X-linked (BMX) with single-digit micromolar IC50s. Several derivatives of 6 were synthesized to gain insight into the binding mode of the compound to the ATP binding pocket. Compound 6 was tested for its effect on anchorage-dependent and independent growth of MDA-MB-231 and MDA-MB-468 breast cancer cells. The effect of 6 on BMX prompted us to evaluate its effect on STAT3 phosphorylation and DNA binding. The compound's inhibition of cell growth led to measurements of survivin, Bcl-XL, p21WAF1/CIP1, and cyclin A2 levels. Finally, LC3B-II levels were quantified following treatment of cells with 6 to determine whether the compound affected autophagy, a process that is known to be activated by STAT3. Compound 6 provides a starting point for the development of small molecules with polypharmacology that can suppress TNBC growth and metastasis.

Small-molecule binding sites to explore protein-protein interactions in the cancer proteome.

The Cancer Genome Atlas (TCGA) offers an unprecedented opportunity to identify small-molecule binding sites on proteins with overexpressed mRNA levels that correlate with poor survival. Here, we analyze RNA-seq and clinical data for 10 tumor types to identify genes that are both overexpressed and correlate with patient survival. Protein products of these genes were scanned for binding sites that possess shape and physicochemical properties that can accommodate small-molecule probes or therapeutic agents (druggable). These binding sites were classified as enzyme active sites (ENZ), protein-protein interaction sites (PPI), or other sites whose function is unknown (OTH). Interestingly, the overwhelming majority of binding sites were classified as OTH. We find that ENZ, PPI, and OTH binding sites often occurred on the same structure suggesting that many of these OTH cavities can be used for allosteric modulation of enzyme activity or protein-protein interactions with small molecules. We discovered several ENZ (PYCR1, QPRT, and HSPA6) and PPI (CASC5, ZBTB32, and CSAD) binding sites on proteins that have been seldom explored in cancer. We also found proteins that have been extensively studied in cancer that have not been previously explored with small molecules that harbor ENZ (PKMYT1, STEAP3, and NNMT) and PPI (HNF4A, MEF2B, and CBX2) binding sites. All binding sites were classified by the signaling pathways to which the protein that harbors them belongs using KEGG. In addition, binding sites were mapped onto structural protein-protein interaction networks to identify promising sites for drug discovery. Finally, we identify pockets that harbor missense mutations previously identified from analysis of TCGA data. The occurrence of mutations in these binding sites provides new opportunities to develop small-molecule probes to explore their function in cancer.

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.

Discovery and characterization of small molecules that target the GTPase Ral.

The Ras-like GTPases RalA and RalB are important drivers of tumour growth and metastasis. Chemicals that block Ral function would be valuable as research tools and for cancer therapeutics. Here we used protein structure analysis and virtual screening to identify drug-like molecules that bind to a site on the GDP-bound form of Ral. The compounds RBC6, RBC8 and RBC10 inhibited the binding of Ral to its effector RALBP1, as well as inhibiting Ral-mediated cell spreading of murine embryonic fibroblasts and anchorage-independent growth of human cancer cell lines. The binding of the RBC8 derivative BQU57 to RalB was confirmed by isothermal titration calorimetry, surface plasmon resonance and (1)H-(15)N transverse relaxation-optimized spectroscopy (TROSY) NMR spectroscopy. RBC8 and BQU57 show selectivity for Ral relative to the GTPases Ras and RhoA and inhibit tumour xenograft growth to a similar extent to the depletion of Ral using RNA interference. Our results show the utility of structure-based discovery for the development of therapeutics for Ral-dependent cancers.

Exploring a structural protein-drug interactome for new therapeutics in lung cancer.

The pharmacology of drugs is often defined by more than one protein target. This property can be exploited to use approved drugs to uncover new targets and signaling pathways in cancer. Towards enabling a rational approach to uncover new targets, we expand a structural protein-ligand interactome () by scoring the interaction among 1000 FDA-approved drugs docked to 2500 pockets on protein structures of the human genome. This afforded a drug-target network whose properties compared favorably with previous networks constructed using experimental data. Among drugs with the highest degree and betweenness two are cancer drugs and one is currently used for treatment of lung cancer. Comparison of predicted cancer and non-cancer targets reveals that the most cancer-specific compounds were also the most selective compounds. Analysis of compound flexibility, hydrophobicity, and size showed that the most selective compounds were low molecular weight fragment-like heterocycles. We use a previously-developed screening approach using the cancer drug erlotinib as a template to screen other approved drugs that mimic its properties. Among the top 12 ranking candidates, four are cancer drugs, two of them kinase inhibitors (like erlotinib). Cellular studies using non-small cell lung cancer (NSCLC) cells revealed that several drugs inhibited lung cancer cell proliferation. We mined patient records at the Regenstrief Medical Record System to explore the possible association of exposure to three of these drugs with occurrence of lung cancer. Preliminary in vivo studies using the non-small cell lung cancer (NCLSC) xenograft model showed that losartan- and astemizole-treated mice had tumors that weighed 50 (p < 0.01) and 15 (p < 0.01) percent less than the treated controls. These results set the stage for further exploration of these drugs and to uncover new drugs for lung cancer therapy.

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-molecule inhibition of the uPAR·uPA interaction: synthesis, biochemical, cellular, in vivo pharmacokinetics and efficacy studies in breast cancer metastasis.

The uPAR·uPA protein-protein interaction (PPI) is involved in signaling and proteolytic events that promote tumor invasion and metastasis. A previous study had identified 4 (IPR-803) from computational screening of a commercial chemical library and shown that the compound inhibited uPAR·uPA PPI in competition biochemical assays and invasion cellular studies. Here, we synthesize 4 to evaluate in vivo pharmacokinetic (PK) and efficacy studies in a murine breast cancer metastasis model. First, we show, using fluorescence polarization and saturation transfer difference (STD) NMR, that 4 binds directly to uPAR with sub-micromolar affinity of 0.2 µM. We show that 4 blocks invasion of breast MDA-MB-231, and inhibits matrix metalloproteinase (MMP) breakdown of the extracellular matrix (ECM). Derivatives of 4 also inhibited MMP activity and blocked invasion in a concentration-dependent manner. Compound 4 also impaired MDA-MB-231 cell adhesion and migration. Extensive in vivo PK studies in NOD-SCID mice revealed a half-life of nearly 5h and peak concentration of 5 µM. Similar levels of the inhibitor were detected in tumor tissue up to 10h. Female NSG mice inoculated with highly malignant TMD-MDA-MB-231 in their mammary fat pads showed that 4 impaired metastasis to the lungs with only four of the treated mice showing severe or marked metastasis compared to ten for the untreated mice. Compound 4 is a promising template for the development of compounds with enhanced PK parameters and greater efficacy.