Direct interaction of TrkA/CD44v3 is essential for NGF-promoted aggressiveness of breast cancer cells

Background CD44 is a multifunctional membrane glycoprotein. Through its heparan sulfate chain, CD44 presents growth factors to their receptors. We have shown that CD44 and Tropomyosin kinase A (TrkA) form a complex following nerve growth factor (NGF) induction. Our study aimed to understand how CD44 and TrkA interact and the consequences of inhibiting this interaction regarding the pro-tumoral effect of NGF in breast cancer. Methods After determining which CD44 isoforms (variants) are involved in forming the TrkA/CD44 complex using proximity ligation assays, we investigated the molecular determinants of this interaction. By molecular modeling, we isolated the amino acids involved and confirmed their involvement using mutations. A CD44v3 mimetic peptide was then synthesized to block the TrkA/CD44v3 interaction. The effects of this peptide on the growth, migration and invasion of xenografted triple-negative breast cancer cells were assessed. Finally, we investigated the correlations between the expression of the TrkA/CD44v3 complex in tumors and histo-pronostic parameters. Results We demonstrated that isoform v3 (CD44v3), but not v6, binds to TrkA in response to NGF stimulation. The final 10 amino acids of exon v3 and the TrkA H112 residue are necessary for the association of CD44v3 with TrkA. Functionally, the CD44v3 mimetic peptide impairs not only NGF-induced RhoA activation, clonogenicity, and migration/invasion of breast cancer cells in vitro but also tumor growth and metastasis in a xenograft mouse model. We also detected TrkA/CD44v3 only in cancerous cells, not in normal adjacent tissues. Conclusion Collectively, our results suggest that blocking the CD44v3/TrkA interaction can be a new therapeutic option for triple-negative breast cancers

Microscale Thermophoresis as a Tool to Study Protein Interactions and Their Implication in Human Diseases

The review highlights how protein–protein interactions (PPIs) have determining roles in most life processes and how interactions between protein partners are involved in various human diseases. The study of PPIs and binding interactions as well as their understanding, quantification and pharmacological regulation are crucial for therapeutic purposes. Diverse computational and analytical methods, combined with high-throughput screening (HTS), have been extensively used to characterize multiple types of PPIs, but these procedures are generally laborious, long and expensive. Rapid, robust and efficient alternative methods are proposed, including the use of Microscale Thermophoresis (MST), which has emerged as the technology of choice in drug discovery programs in recent years. This review summarizes selected case studies pertaining to the use of MST to detect therapeutically pertinent proteins and highlights the biological importance of binding interactions, implicated in various human diseases. The benefits and limitations of MST to study PPIs and to identify regulators are discussed

A Potential Off-Target Effect of the Wnt/β-Catenin Inhibitor KYA1797K: PD-L1 Binding and Checkpoint Inhibition

Introduction: The quest for small molecule inhibitors of the PD-1/PD-L1 checkpoint continues in parallel to the extensive development of monoclonal antibodies directed against this immune checkpoint. Drug screening strategies are being set up to identify novel PD-L1 inhibitors.Methods: A virtual screening based on molecular docking with the PD-L1 protein dimer has been performed to identify a new binder. Binding of the identified ligand to PD-L1 has been validated experimentally using a microscale thermophoresis (MST) assay. The cellular effect of the compound was evidenced using a fluorescence resonance energy transfer (FRET) assay based on activation of tyrosine phosphatase SHP-2.Results: We have identified the potent Wnt/β-catenin inhibitor KYA1797K as a weak PD-L1 binder. Molecular docking suggested that the compound can bind to the interface of a PD-L1 dimer, with a geometry superimposable to that of the reference PD-L1 inhibitor BMS-202. The atypical 2-thioxo-4-thiazolidinone motif of KYA1797K, derived from the natural product rhodanine, plays a major role in the interaction with PD-L1. Binding of KYA1797K to recombinant hPD-L1 was validated experimentally, using MST. The drug was found to bind modestly but effectively to hPD-L1. The FRET assay confirmed the weak capacity of KYA1797K to interfere with the activation of SHP-2 upon its interaction with human PD-1.Discussion: Collectively, the data show that KYA1797K could function as a weak modulator of the PD-1/PD-L1 checkpoint. This effect may contribute, at least partially, to the reported capacity of the β-catenin inhibitor to downregulate PD-L1 in cancer cells. The work also underlines the interest to further consider the rhodanine moiety as a chemical motif for the design of new PD-L1 binders

Measurement of Protein-Protein Interactions through Microscale Thermophoresis (MST)

The binding interactions of PD-1 and PD-L1 have been studied by surface plasmonresonance (SPR) and isothermal titration calorimetry (ITC) over the past few years, but theseinvestigations resulted in controversy regarding the values of the dissociation constant (Kd) (Freeman et al., 2000). MST is a powerful new method for the quantitative analysis of protein-protein interactions (PPIs) with low sample consumption. The technique is based on the movement of molecules along microscopic temperature gradients, and it detects changes in their hydration shell, charge or size. One binding partner is fluorescently labeled, while the other binding partner remains label-free. We used a protocol that allows the determination of the binding affinity by MST without purification of the target protein from the cell lysate. The application of this MST method to PD-1-eGFP and PD-L1-eGFP expressed in CHO-K1 cells allowed us, for the first time, to determine the affinity of the complex formed between PD-1 and its ligand PD-L1 during tumor escape. The protocol has a variety of potential applications for studying the interactions of proteins with small molecules

Drug Repurposing to Enhance Antitumor Response to PD-1/PD-L1 Immune Checkpoint Inhibitors

Monoclonal antibodies targeting the PD-1/PD-L1 immune checkpoint have considerably improved the treatment of some cancers, but novel drugs, new combinations, and treatment modalities are needed to reinvigorate immunosurveillance in immune-refractory tumors. An option to elicit antitumor immunity against cancer consists of using approved and marketed drugs known for their capacity to modulate the expression and functioning of the PD-1/PD-L1 checkpoint. Here, we have reviewed several types of drugs known to alter the checkpoint, either directly via the blockade of PD-L1 or indirectly via an action on upstream effectors (such as STAT3) to suppress PD-L1 transcription or to induce its proteasomal degradation. Specifically, the repositioning of the approved drugs liothyronine, azelnidipine (and related dihydropyridine calcium channel blockers), niclosamide, albendazole/flubendazole, and a few other modulators of the PD-1/PD-L1 checkpoint (repaglinide, pimozide, fenofibrate, lonazolac, propranolol) is presented. Their capacity to bind to PD-L1 or to repress its expression and function offer novel perspectives for combination with PD-1 targeted biotherapeutics. These known and affordable drugs could be useful to improve the therapy of cancer

Antioxidant Properties and Aldehyde Reactivity of PD-L1 Targeted Aryl-Pyrazolone Anticancer Agents

International audienceSmall molecules targeting the PD-1/PD-L1 checkpoint are actively searched to complement the anticancer arsenal. Different molecular scaffolds have been reported, including phenyl-pyrazolone derivatives which potently inhibit binding of PD-L1 to PD-1. These molecules are structurally close to antioxidant drug edaravone (EDA) used to treat amyotrophic lateral sclerosis. For this reason, we investigated the capacity of five PD-L1-binding phenyl-pyrazolone compounds (1–5) to scavenge the formation of oxygen free radicals using electron spin resonance spectroscopy with DPPH/DMPO probes. In addition, the reactivity of the compounds toward the oxidized base 5-formyluracil (5fU) was assessed using chromatography coupled to mass spectrometry and photodiode array detectors. The data revealed that the phenyl-pyrazolone derivatives display antioxidant properties and exhibit a variable reactivity toward 5fU. Compound 2 with a N-dichlorophenyl-pyrazolone moiety cumulates the three properties, being a potent PD-L1 binder, a robust antioxidant and an aldehyde-reactive compound. On the opposite, the adamantane derivative 5 is a potent PD-L1 binding with a reduced antioxidant potential and no aldehyde reactivity. The nature of the substituent on the phenyl-pyrazolone core modulates the antioxidant capacity and reactivity toward aromatic aldehydes. The molecular signature of the compound can be adapted at will, to confer additional properties to these PD-L1 binders

Novel PD-L1-Targeted Phenyl-Pyrazolone Derivatives with Antioxidant Properties

Orally-active anticancer small molecules targeting the PD-1/PD-L1 immune checkpoint are actively searched. Phenyl-pyrazolone derivatives with a high affinity for PD-L1 have been designed and characterized. In addition, the phenyl-pyrazolone unit acts as a scavenger of oxygen free radicals, providing antioxidant effects. The mechanism is known for the drug edaravone (1) which is also an aldehyde-reactive molecule. The present study reports the synthesis and functional characterization of new molecules (2–5) with an improved anti-PD-L1 activity. The leading fluorinated molecule 5 emerges as a potent checkpoint inhibitor, avidly binding to PD-L1, inducing its dimerization, blocking PD-1/PD-L1 signaling mediated by phosphatase SHP-2 and reactivating the proliferation of CTLL-2 cells in the presence of PD-L1. In parallel, the compound maintains a significant antioxidant activity, characterized using electron paramagnetic resonance (EPR)-based free radical scavenging assays with the probes DPPH and DMPO. The aldehyde reactivity of the molecules was investigated using 4-hydroxynonenal (4-HNE), which is a major lipid peroxidation product. The formation of drug-HNE adducts, monitored by high resolution mass spectrometry (HRMS), was clearly identified and compared for each compound. The study leads to the selection of compound 5 and the dichlorophenyl-pyrazolone unit as a scaffold for the design of small molecule PD-L1 inhibitors endowed with antioxidant properties

MUC4-ErbB2 Oncogenic Complex: Binding studies using Microscale Thermophoresis

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