Antitumor Activity and Mechanistic Insights of a Mitochondria-Targeting Cu(I) Complex.

Using copper-ionophores to translocate extracellular copper into mitochondria is a clinically validated anticancer strategy that has been identified as a new type of regulated cell death termed "cuproptosis." This study reports a mitochondria-targeting Cu(I) complex, Cu(I)Br(PPh3)3 (CBP), consisting of a cuprous ion coordinated by three triphenylphosphine moieties and a Br atom. CBP exhibited antitumor and antimetastatic efficacy in vitro and in vivo by specifically targeting mitochondria instigating mitochondrial dysfunction. The cytotoxicity of CBP could only be reversed by a copper chelator rather than inhibitors of the known cell death, indicating copper-dependent cytotoxicity. Furthermore, CBP induced the oligomerization of lipoylated proteins and the loss of Fe-S cluster proteins, consistent with characteristic features of cuproptosis. Additionally, CBP induced remarkable intracellular generation of reactive oxygen species (ROS) through a Fenton-like reaction, indicating a complex antitumor mechanism. This is a proof-of-concept study exploiting the antitumor activity and mechanism of the Cu(I)-based mitochondria-targeting therapy.

Engineered protein cages with enhanced extracellular drug release for elevated antitumor efficacy.

Human heavy chain ferritin (HFn) protein cage has been explored as a nanocarrier for targeted anticancer drug delivery. Here, we introduced a matrix metalloproteinases (MMPs)-cleavable sequence into the DE loop of HFn, creating an MMP-responsive variant, MR-HFn, for localized and extracellular drug release. The crystal structure of MR-HFn revealed that the addition of the MMPs recognition sequence did not affect the self-assembly of HFn but presented a surface-exposed loop susceptible to MMPs cleavage. Biochemical analysis indicated that this engineered protein cage is responsive to MMPs, enabling the targeted release of encapsulated drugs. To evaluate the therapeutic potential of this engineered protein cage, monosubstituted ß-carboxy phthalocyanine zinc (CPZ), a type of photosensitizer, was loaded inside this protein cage. The prepared CPZ@MR-HFn showed higher uptake and stronger phototoxicity in MMPs overexpressed tumor cells, as well as enhanced penetration into multicellular tumor spheroids compared with its counterpart CPZ@HFn in vitro. In vivo, CPZ@MR-HFn displayed a higher tumor inhibitory rate than CPZ@HFn under illumination. These results indicated that MR-HFn is a promising nanocarrier for anticancer drug delivery and the MMP-responsive strategy here can also be adapted for other stimuli.

A low bleeding risk thrombolytic agent: citPA5.

AIMS: Alteplase is a cornerstone thrombolytic agent in clinical practice, but presents a potential bleeding risk. Stroke patients need pre-screening to exclude hemorrhagic stroke before using Alteplase. In this study, we develop a new thrombolytic agent citPA5, characterized by an enhanced safety profile and minimal bleeding tendency. METHODS AND RESULTS: A clot lysis agent, named citPA5, is developed based on rtPA with point mutations to completely suppress its proteolytic activity in the absence of fibrin. In the presence of fibrin, citPA5 exhibited significantly higher fibrinolytic activity (a 15.8-fold increase of kcat/Km). Furthermore, citPA5 showed resistance to endogenous fibrinolysis inhibitor, PAI-1, resulting in enhanced potency. In a series of safety evaluation experiments, including thrombelastography (TEG) assay, mice tail bleeding assay, and a murine intracerebral hemorrhage (ICH) model, citPA5 did not cause systemic bleeding or worsen intracerebral hemorrhage compared to Alteplase. This highlights the low risk of bleeding associated with citPA5. Finally, we found that citPA5 effectively improved cerebral blood flow and reduced infarct volume in a carotid embolism-induced stroke (CES) model. CONCLUSIONS: This clot lysis agent, citPA5, not only exhibits a low risk of bleeding but also demonstrates highly effective thrombolysis capabilities. As a result, citPA5 shows great potential for administration prior to the classification of stroke types, making it possible for use in ambulances at the onset of stroke when symptoms are identified. The findings presented in this study also suggest that this strategy could be applied to develop a new generation of fibrinolytic drugs that offer greater safety and specificity in targeting fibrin.

Endoplasmic Reticulum Protein 72 Regulates Integrin Mac-1 Activity to Influence Neutrophil Recruitment.

BACKGROUND: Integrins mediate the adhesion, crawling, and migration of neutrophils during vascular inflammation. Thiol exchange is important in the regulation of integrin functions. ERp72 (endoplasmic reticulum-resident protein 72) is a member of the thiol isomerase family responsible for the catalysis of disulfide rearrangement. However, the role of ERp72 in the regulation of Mac-1 (integrin αMß2) on neutrophils remains elusive. METHODS: Intravital microscopy of the cremaster microcirculation was performed to determine in vivo neutrophil movement. Static adhesion, flow chamber, and flow cytometry were used to evaluate in vitro integrin functions. Confocal fluorescent microscopy and coimmunoprecipitation were utilized to characterize the interactions between ERp72 and Mac-1 on neutrophil surface. Cell-impermeable probes and mass spectrometry were used to label reactive thiols and identify target disulfide bonds during redox exchange. Biomembrane force probe was performed to quantitatively measure the binding affinity of Mac-1. A murine model of acute lung injury induced by lipopolysaccharide was utilized to evaluate neutrophil-associated vasculopathy. RESULTS: ERp72-deficient neutrophils exhibited increased rolling but decreased adhesion/crawling on inflamed venules in vivo and defective static adhesion in vitro. The defect was due to defective activation of integrin Mac-1 but not LFA-1 (lymphocyte function-associated antigen-1) using blocking or epitope-specific antibodies. ERp72 interacted with Mac-1 in lipid rafts on neutrophil surface leading to the reduction of the C654-C711 disulfide bond in the αM subunit that is critical for Mac-1 activation. Recombinant ERp72, via its catalytic motifs, increased the binding affinity of Mac-1 with ICAM-1 (intercellular adhesion molecule-1) and rescued the defective adhesion of ERp72-deficient neutrophils both in vitro and in vivo. Deletion of ERp72 in the bone marrow inhibited neutrophil infiltration, ameliorated tissue damage, and increased survival during murine acute lung injury. CONCLUSIONS: Extracellular ERp72 regulates integrin Mac-1 activity by catalyzing disulfide rearrangement on the αM subunit and may be a novel target for the treatment of neutrophil-associated vasculopathy.

Specific inhibition on PAI-1 reduces the dose of Alteplase for ischemic stroke treatment.

Administration of recombinant tPA (rtPA, or trade name Alteplase®) is an FDA-approved therapy for acute ischemic stroke (AIS), but poses the risk of hemorrhagic complications. Recombinant tPA can be rapidly inactivated by the endogenous inhibitor, plasminogen activator inhibitor 1 (PAI-1). In this work, we study a novel treatment approach that combines a PAI-1 inhibitor, PAItrap4, with a reduced dose of rtPA to address the hemorrhagic concern of rtPA. PAItrap4 is a highly specific and very potent protein-based inhibitor of PAI-1, comprising of a variant of uPA serine protease domain, human serum albumin, and a cyclic RGD peptide. PAItrap4 efficiently targets and inhibits PAI-1 on activated platelets, and also possesses a long half-life in vivo. Our results demonstrate that PAItrap4 effectively counteracts the inhibitory effects of PAI-1 on rtPA, preserving rtPA activity based on amidolytic and clot lysis assays. In an in vivo murine stroke model, PAItrap4, together with low-dose rtPA, enhances the blood perfusion in the stroke-affected areas, reduces infarct size, and promotes neurological recovery in mice. Importantly, such treatment does not increase the amount of cerebral hemorrhage, thus reducing the risk of cerebral hemorrhage. In addition, PAItrap4 does not compromise the normal blood coagulation function in mice, demonstrating its safety as a therapeutic agent. These findings highlight this combination therapy as a promising alternative for the treatment of ischemic stroke, offering improved safety and efficacy.

In silico screening of natural products as uPAR inhibitors via multiple structure-based docking and molecular dynamics simulations.

Cancer remains one of the most pressing challenges to global healthcare, exerting a significant impact on patient life expectancy. Cancer metastasis is a critical determinant of the lethality and treatment resistance of cancer. The urokinase-type plasminogen activator receptor (uPAR) shows great potential as a target for anticancer and antimetastatic therapies. In this work, we aimed to identify potential uPAR inhibitors by structural dynamics-based virtual screenings against a natural product library on four representative apo-uPAR structural models recently derived from long-timescale molecular dynamics (MD) simulations. Fifteen potential inhibitors (NP1-NP15) were initially identified through molecular docking, consensus scoring, and visual inspection. Subsequently, we employed MD-based molecular mechanics-generalized Born surface area (MM-GBSA) calculations to evaluate their binding affinities to uPAR. Structural dynamics analyses further indicated that all of the top 6 compounds exhibited stable binding to uPAR and interacted with the critical residues in the binding interface between uPAR and its endogenous ligand uPA, suggesting their potential as uPAR inhibitors by interrupting the uPAR-uPA interaction. We finally predicted the ADMET properties of these compounds. The natural products NP5, NP12, and NP14 with better binding affinities to uPAR than the uPAR inhibitors previously discovered by us were proven to be potentially orally active in humans. This work offers potential uPAR inhibitors that may contribute to the development of novel effective anticancer and antimetastatic therapeutics.Communicated by Ramaswamy H. Sarma.

Structural and Functional Insights into the Stealth Protein CpsY of Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) is an important and harmful intracellular pathogen that is responsible for the cause of tuberculosis (TB). Mtb capsular polysaccharides can misdirect the host's immune response pathways, resulting in additional challenges in TB treatment. These capsule polysaccharides are biosynthesized by stealth proteins, including CpsY. The structure and functional mechanism of Mtb CpsY are not completely delineated. Here, we reported the crystal structure of CpsY201-520 at 1.64 Å. CpsY201-520 comprises three ß-sheets with five α-helices on one side and three on the other. Four conserved regions (CR1-CR4) are located near and at the base of its catalytic cavity, and three spacer segments (S1-S3) surround the catalytic cavity. Site-directed mutagenesis demonstrated the strict conservation of R419 at CR3 and S1-S3 in regulating the phosphotransferase activity of CpsY201-520. In addition, deletion of S2 or S3 (∆S2 or ∆S3) dramatically increased the activity compared to the wild-type (WT) CpsY201-520. Results from molecular dynamics (MD) simulations showed that S2 and S3 are highly flexible. Our study provides new insights for the development of new vaccines and targeted immunotherapy against Mtb.

Potential Roles and Future Perspectives of Chitinase 3-like 1 in Macrophage Polarization and the Development of Diseases.

Chitinase-3-like protein 1 (CHI3L1), a chitinase-like protein family member, is a secreted glycoprotein that mediates macrophage polarization, inflammation, apoptosis, angiogenesis, and carcinogenesis. Abnormal CHI3L1 expression has been associated with multiple metabolic and neurological disorders, including diabetes, atherosclerosis, and Alzheimer's disease. Aberrant CHI3L1 expression is also reportedly associated with tumor migration and metastasis, as well as contributions to immune escape, playing important roles in tumor progression. However, the physiological and pathophysiological roles of CHI3L1 in the development of metabolic and neurodegenerative diseases and cancer remain unclear. Understanding the polarization relationship between CHI3L1 and macrophages is crucial for disease progression. Recent research has uncovered the complex mechanisms of CHI3L1 in different diseases, highlighting its close association with macrophage functional polarization. In this article, we review recent findings regarding the various disease types and summarize the relationship between macrophages and CHI3L1. Furthermore, this article also provides a brief overview of the various mechanisms and inhibitors employed to inhibit CHI3L1 and disrupt its interaction with receptors. These endeavors highlight the pivotal roles of CHI3L1 and suggest therapeutic approaches targeting CHI3L1 in the development of metabolic diseases, neurodegenerative diseases, and cancers.

Crystal structures of human serum albumin in complex with lysophosphatidylcholine.

Lysophospholipids (lysoPLs) are crucial metabolites involved in various physiological and pathological cellular processes. Understanding their binding interactions, particularly with human serum albumin (HSA), is essential due to their role in regulating lysoPLs-induced cytotoxicity. However, the precise mechanism of lysoPLs binding to HSA remains elusive. In this study, we employed fluorescence quenching and optical interferometry assays to demonstrate direct binding between lysophosphatidylcholine (LPC) and HSA (KD = 25 µM). Furthermore, we determined crystal structures of HSA in complex with LPC, both in the absence and the presence of the endogenous fatty acid myristate (14:0). The crystal structure of binary HSA:LPC revealed that six LPC molecules are bound to HSA at the primary fatty acid binding sites. Interestingly, the ternary HSA:Myr:LPC structure demonstrated the continued binding of three LPC molecules to HSA at binding sites 1, 3, and 5 in the presence of myristate. These findings support HSA's role as a carrier protein for lysoPLs in blood plasma and provide valuable insights into the structural basis of their binding mechanisms.

From disinfectants to antibiotics: Enhanced biosafety of quaternary ammonium compounds by chemical modification.

The excessive use of quaternary ammonium compounds (QACs) following the COVID-19 pandemic has raised substantial concerns regarding their biosafety. Overuse of QACs has been associated with chronic biological adverse effects, including genotoxicity or carcinogenicity. In particular, inadvertent intravascular administration or oral ingestion of QACs can lead to fatal acute toxicity. To enhance the biosafety and antimicrobial efficacy of QACs, this study reports a new series of QACs, termed as PACs, with the alkyl chain of benzalkonium substituted by a phthalocyanine moiety. Firstly, the rigid phthalocyanine moiety enhances the selectivity of QACs to bacteria over human cells and reduces alkyl chain's entropic penalty of binding to bacterial membranes. Furthermore, phthalocyanine neutralizes hemolysis and cytotoxicity of QACs by binding with albumin in plasma. Our experimental results demonstrate that PACs inherit the optical properties of phthalocyanine and validate the broad-spectrum antibacterial activity of PACs in vitro. Moreover, the intravascular administration of the most potent PAC, PAC1a, significantly reduced bacterial burden and ameliorated inflammation level in a bacteria-induced septic mouse model. This study presents a new strategy to improve the antimicrobial efficacy and biosafety of QACs, thus expanding their range of applications to the treatment of systemic infections.

Polyphosphate as an antithrombotic target and hemostatic agent.

Polyphosphate (PolyP) is a polymer comprised of linear phosphate units connected by phosphate anhydride bonds. PolyP exists in a diverse range of eukaryotes and prokaryotes with varied chain lengths ranging from six to thousands of phosphate units. Upon activation, human platelets and neutrophils release short-chain PolyP, along with other components, to initiate the coagulation pathway. Long-chain PolyP derived from cellular or bacterial organelles exhibits higher proinflammatory and procoagulant effects compared to short-chain PolyP. Notably, PolyP has been identified as a low-hemorrhagic antithrombotic target since neutralizing plasma PolyP suppresses the thrombotic process without impairing the hemostatic functions. As an inorganic polymer without uniform steric configuration, PolyP is typically targeted by cationic polymers or recombinant polyphosphatases rather than conventional antibodies, small-molecule compounds, or peptides. Additionally, because of its procoagulant property, PolyP has been incorporated in wound-dressing materials to facilitate blood hemostasis. This review summarizes current studies on PolyP as a low-hemorrhagic antithrombotic target and the development of hemostatic materials based on PolyP.

Exploring the mechanism of photosensitizer conjugation on membrane perturbation of antimicrobial peptide: A multiscale molecular simulation study.

Antimicrobial peptides (AMPs) exert their biological functions by perturbation with cellular membrane. Conjugation of AMPs with photosensitizer (PS) is a promising strategy for enhancing the efficacy and reducing systemic toxicity of AMPs. However, it is still elusive how the conjugated PS impacts the perturbation of AMPs on cell membrane from molecular level. Here, we addressed this issue by a multiscale computational strategy on pyropheophorbide-a (PPA) conjugated K6L9 (PPA-K6L9), a PS-AMP conjugate developed by us previously. Our atomistic molecular dynamics (MD) simulations revealed that the porphyrin moiety of PPA enhanced the stability of the conjugate in a lipid bilayer membrane model. Moreover, such moiety also maintained the amphipathic structure of K6L9, which is crucial for membrane pore formation. Coarse-grained MD simulations further showed that the conjugates aggregated in membrane environment and formed more stable toroidal pores with respect to K6L9 alone, suggesting the conjugation of PPA may enhance the membrane-disruption activity of K6L9. Consistent with this, our cellular experiments confirmed that PPA-K6L9 was more toxic to 4 T1 tumor cells than K6L9. This study provides insights into the mechanism by which PS-AMP conjugates disrupt cellular membranes and could aid in the design of more potent AMP conjugates.

Monomer and Oligomer Transition of Zinc Phthalocyanine Is Key for Photobleaching in Photodynamic Therapy.

Photodynamic therapy (PDT) is recognized as a powerful method to inactivate cells. However, the photosensitizer (PS), a key component of PDT, has suffered from undesired photobleaching. Photobleaching reduces reactive oxygen species (ROS) yields, leading to the compromise of and even the loss of the photodynamic effect of the PS. Therefore, much effort has been devoted to minimizing photobleaching in order to ensure that there is no loss of photodynamic efficacy. Here, we report that a type of PS aggregate showed neither photobleaching nor photodynamic action. Upon direct contact with bacteria, the PS aggregate was found to fall apart into PS monomers and thus possessed photodynamic inactivation against bacteria. Interestingly, the disassembly of the bound PS aggregate in the presence of bacteria was intensified by illumination, generating more PS monomers and leading to an enhanced antibacterial photodynamic effect. This demonstrated that on a bacterial surface, the PS aggregate photo-inactivated bacteria via PS monomer during irradiation, where the photodynamic efficiency was retained without photobleaching. Further mechanistic studies showed that PS monomers disrupted bacterial membranes and affected the expression of genes related to cell wall synthesis, bacterial membrane integrity, and oxidative stress. The results obtained here are applicable to other types of PSs in PDT.

Dental Bleaching with Phthalocyanine Photosensitizers: Effects on Dentin Color and Collagen Content.

With the increasing demand for tooth bleaching in esthetic dentistry, its safety has been the focus of a comprehensive body of literature. In this context, the aim of the present study was to evaluate the application effects of pentalysine ß-carbonylphthalocyanine zinc (ZnPc(Lys)5)-mediated photodynamic therapy in dentin bleaching and its effects on dentin collagen. We first established a new and reproducible tooth staining model using dentin blocks stained by Orange II and then bleached with ZnPc(Lys)5 (25 µM) and hydrogen peroxide (10% or 30%). Data were analyzed with one- and two-way ANOVA and a significance level of p < 0.05. ZnPc(Lys)5 effectively bleached the dentin samples to an extent comparable to hydrogen peroxide at either 10% or 30% concentrations. Further studies on the dentin morphology, chemical element distribution, and protein constituents, using an electron microscope, energy dispersive spectroscopy, X-ray photoelectron spectroscopy, and SDS-PAGE, demonstrated that treatment with the photosensitizer preserved the dentin structure and, at the same time, the major organic component, collagen type I. For comparison, hydrogen peroxide (10% or 30%) treatment significantly degraded the collagen protein. This work indicated that the photosensitizer exerts potent bleaching effects on dentin staining; importantly, does not damage dentin and its collagen content; and opens up a new strategy to further explore various photosensitizers for the bleaching of both tooth enamel and dentin.

A Serum-Stable supramolecular drug carrier for chemotherapeutics fabricated by a Peptide-Photosensitizer conjugate.

Supramolecular assemblies fabricated by peptide-photosensitizer conjugates have attracted increasing attentions in recent years as drug carriers for chemotherapeutics (CTs). However, these assemblies have been known to suffer from disintegration by serum components leading to off-target drug release, and thereby impairing antitumor effects and causing systemic toxicities. To address this problem, this study reports a nano-architectural self-assembly peptide-photosensitizer carrier (NSPC) fabricated by conjugating a phthalocyanine derivative (MCPZnPc) and ε-poly-l-lysine (EPL). By engineering the core and peripheral interactions, MCPZnPC-EPL (M-E) NSPC firmly encapsulated multiple CTs, creating CT@M-E NSPCs that were highly stable against disintegration in serum. More importantly, CT@M-E NSPCs exhibited controlled release of CTs in tumor tissues. The antitumor effects of CTs were further promoted by the synergism with the reactivated photodynamic effect. Furthermore, M-E NSPC-encapsulation optimized CTs' biodistribution reducing adverse effects in vivo. This study provides a serum-stable supramolecular drug delivery system with photodynamic effect, which is applicable for a broad-range of CTs to promote antitumor effects and ameliorate adverse effects.

Enhanced inhibition of protein disulfide isomerase and anti-thrombotic activity of a rutin derivative: rutin:Zn complex.

Rutin is a flavonoid that exists in plants and in commonly consumed foods. In recent years, rutin has been demonstrated to have anti-thrombotic efficacy through its inhibition of protein disulfide isomerase. However, the low aqueous solubility and high dose limit the therapeutic applications of rutin. In this study, we found that the chelation of zinc ions increased rutin aqueous solubility by 4-fold. More importantly, the thus-formed rutin:Zn complex inhibited PDI activity more potently than rutin itself. In a murine model with electric current-induced arterial thrombosis, the rutin:Zn complex slowed mouse arterial occlusion compared to rutin without increasing bleeding risk. Thus, the zinc chelation not only improved rutin aqueous solubility but achieved stronger inhibition of PDI. Furthermore, zinc chelation of a selected list of flavonoids containing the adjacent keto and phenoxy groups also increased their inhibition of PDI. Hence, our study provides a strategy to promote flavonoids' anti-thrombotic properties.

Cryo-EM structure of the RuvAB-Holliday junction intermediate complex from Pseudomonas aeruginosa.

Holliday junction (HJ) is a four-way structured DNA intermediate in homologous recombination. In bacteria, the HJ-specific binding protein RuvA and the motor protein RuvB together form the RuvAB complex to catalyze HJ branch migration. Pseudomonas aeruginosa (P. aeruginosa, Pa) is a ubiquitous opportunistic bacterial pathogen that can cause serious infection in a variety of host species, including vertebrate animals, insects and plants. Here, we describe the cryo-Electron Microscopy (cryo-EM) structure of the RuvAB-HJ intermediate complex from P. aeruginosa. The structure shows that two RuvA tetramers sandwich HJ at the junction center and disrupt base pairs at the branch points of RuvB-free HJ arms. Eight RuvB subunits are recruited by the RuvA octameric core and form two open-rings to encircle two opposite HJ arms. Each RuvB subunit individually binds a RuvA domain III. The four RuvB subunits within the ring display distinct subdomain conformations, and two of them engage the central DNA duplex at both strands with their C-terminal ß-hairpins. Together with the biochemical analyses, our structure implicates a potential mechanism of RuvB motor assembly onto HJ DNA.

A triple fusion tissue-type plasminogen activator (TriF-ΔtPA) enhanced thrombolysis in carotid embolism-induced stroke model.

Recombinant tissue-type plasminogen activator (rtPA, or Alteplase) is the first approved thrombolytic drug for acute ischemic stroke, but suffers from a short half-life and poor resistance to plasminogen activator inhibitor (PAI-1), limiting its clinical use. The development of novel thrombolytic agents with improved benefit/risk balance has always been of great significance. In this study, we identified a mutant of serine protease domain of tPA (named ΔtPAA146V) capable of escaping the inhibition by endogenous PAI-1 with 66-fold increased resistance compared to the wild type tPA. Based on this mutant, we generated a triple fusion ΔtPA (TriF-ΔtPA) containing albumin and fibrin binding peptide(FBP). The fusion with albumin effectively prolonged the plasma half-life of ΔtPA in mice to 144 min, which is much longer than ΔtPA and did not affect its thrombolytic activity. Furthermore, FBP rendered fibrin specificity of the fusion protein, giving a dissociation constant of âˆ¼ 25 ± 0.9 µM. In a novel murine carotid embolism-induced stroke (CES) model, i.v. administration of TriF-ΔtPA promoted vascular recanalization, reduced infarct volume, and mitigated neurobehavioral deficits more significantly compared to ΔtPA-HSA or Alteplase, showing little bleeding risk. Together, this long-acting PAI-1-resistant thrombolytic agent holds great potential for clinical applications.

Structural Dynamics-Driven Discovery of Anticancer and Antimetastatic Effects of Diltiazem and Glibenclamide Targeting Urokinase Receptor.

Diltiazem and glibenclamide are commonly used hypotensive and antidiabetic drugs. This study reports the discovery of the potential antitumor and antimetastatic effects of these two drugs using a structural dynamics-driven virtual screening targeting urokinase receptor (uPAR). Owing to uPAR's high flexibility, currently resolved crystal structures of uPAR, all in ligand-bound states, provide limited representations of its physiological conformation. To improve the accuracy of screening, we performed a long-timescale molecular dynamics simulation and obtained the representative conformations of apo-uPAR as the targets for our screening. Experimentally, we demonstrated that diltiazem and glibenclamide bound uPAR with KD values in the micromolar range. In addition, both compounds effectively suppressed tumor growth and metastasis in a uPAR-dependent manner in vitro and in vivo. This work not only provides two potent uPAR inhibitors but also reports a proof-of-concept study on the potential off-label antitumor and antimetastatic uses of diltiazem and glibenclamide.

Albumin-based drug carrier targeting urokinase receptor for cancer therapy.

Urokinase plasminogen activator receptor (uPAR) is a key participant in extracellular proteolysis, tissue remodeling and cell motility. uPAR overexpresses in most solid tumors and several hematologic malignancies, but has low levels on normal tissues, thus is advocated as a molecular target for cancer therapy. One of the obstacles for the evaluation of uPAR targeting agents in preclinical study is the species specificity, where targeting agents for human uPAR  usually not bind to murine uPAR. Here, we develop a targeting agent that binds to both murine and human uPAR. This targeting agent is genetically fused to human serum albumin, a commonly used drug carrier, and the final construct is named as uPAR targeting carrier (uPARTC). uPARTC binds specifically to uPAR-overexpressing 293T/huPAR and 293T/muPAR as demonstrated by flow cytometry. A cytotoxic compound, celastrol, is embedded into uPARTC non-covalently. The resulting macromolecular complex show effective proliferation inhibition on both murine and human uPAR overexpressing cells, and exhibit potent antitumor efficacy on hepatoma H22-bearing mice. This work demonstrates that uPARTC is a promising tumor targeting drug carrier, which address the species-specificity challenge of uPAR targeting agents and can be used to load other cytotoxic compounds.