Pyrazolones as inhibitors of immune checkpoint blocking the PD-1/PD-L1 interaction.

Immuno-therapy has become a leading strategy to fight cancer. Over the past few years, immuno-therapies using checkpoint inhibitor monoclonal antibodies (mAbs) against programmed death receptor 1 (PD-1) and programmed death ligand 1 (PD-L1) have demonstrated improved survival compared with chemotherapy. We describe the microwave-assisted synthesis and the characterization of an innovative series of synthetic compounds endowed with nanomolar activity against PD-L1. The properties of the compounds were characterized using several biophysical techniques including microscale thermophoresis (MST) and fluorescence resonance energy transfer (FRET) measurements. A few small molecules demonstrated a high affinity for human PD-L1, potently disrupted the PD-L1:PD-1 interaction and inhibited Src homology region 2 domain-containing phosphatase (SHP2) recruitment to PD-1. More than 30 molecules from the pyrazolone family have been synthesized and 5 highly potent "PD-L1 silencing compounds" have been identified, based on in vitro measurements. Structure-activity relationships have been defined and ADME properties were evaluated. The phenyl-pyrazolone unit offers novel perspectives to design PD-L1-targeting agents, potentially useful to combat cancer and other pathologies implicating the PD-1/PD-L1 checkpoint.

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

The binding interactions of PD-1 and PD-L1 have been studied by surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) over the past few years, but these investigations 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.

Lactate Dehydrogenase B Controls Lysosome Activity and Autophagy in Cancer.

Metabolic adaptability is essential for tumor progression and includes cooperation between cancer cells with different metabolic phenotypes. Optimal glucose supply to glycolytic cancer cells occurs when oxidative cancer cells use lactate preferentially to glucose. However, using lactate instead of glucose mimics glucose deprivation, and glucose starvation induces autophagy. We report that lactate sustains autophagy in cancer. In cancer cells preferentially to normal cells, lactate dehydrogenase B (LDHB), catalyzing the conversion of lactate and NAD(+) to pyruvate, NADH and H(+), controls lysosomal acidification, vesicle maturation, and intracellular proteolysis. LDHB activity is necessary for basal autophagy and cancer cell proliferation not only in oxidative cancer cells but also in glycolytic cancer cells.

A mitochondrial switch promotes tumor metastasis.

Metastatic progression of cancer is associated with poor outcome, and here we examine metabolic changes underlying this process. Although aerobic glycolysis is known to promote metastasis, we have now identified a different switch primarily affecting mitochondria. The switch involves overload of the electron transport chain (ETC) with preserved mitochondrial functions but increased mitochondrial superoxide production. It provides a metastatic advantage phenocopied by partial ETC inhibition, another situation associated with enhanced superoxide production. Both cases involved protein tyrosine kinases Src and Pyk2 as downstream effectors. Thus, two different events, ETC overload and partial ETC inhibition, promote superoxide-dependent tumor cell migration, invasion, clonogenicity, and metastasis. Consequently, specific scavenging of mitochondrial superoxide with mitoTEMPO blocked tumor cell migration and prevented spontaneous tumor metastasis in murine and human tumor models.