Targeted protein degradation modulates protein function beyond the inhibition of enzyme activity or protein–protein interactions. Most degrader drugs function by directly mediating the proximity between a neosubstrate and a hijacked E3 ligase. Here we identify pseudo-natural products derived from (−)-myrtanol, termed iDegs, that inhibit and induce degradation of the immunomodulatory enzyme indoleamine-2,3-dioxygenase 1 (IDO1) by a distinct mechanism. iDegs boost IDO1 ubiquitination and degradation by the cullin-RING E3 ligase CRL2 ^KLHDC3 , which we identified to natively mediate ubiquitin-mediated degradation of IDO1. Therefore, iDegs increase IDO1 turnover using the native proteolytic pathway. In contrast to clinically explored IDO1 inhibitors, iDegs reduce the formation of kynurenine by both inhibition and induced degradation of the enzyme and thus also modulate the non-enzymatic functions of IDO1. This unique mechanism of action may open up alternative therapeutic opportunities for the treatment of cancer beyond classical inhibition of IDO1.

Indoleamine 2,3-dioxygenase 2 (IDO2) is a paralog of Indoleamine 2,3-dioxygenase 1 (IDO1), a tryptophan-degrading enzyme producing immunomodulatory molecules. However, the two proteins are unlikely to carry out the same functions. IDO2 shows little or no tryptophan catabolic activity and exerts contrasting immunomodulatory roles in a context-dependent manner in cancer and autoimmune diseases. The recently described potential non-enzymatic activity of IDO2 has suggested its possible involvement in alternative pathways, resulting in either pro- or anti-inflammatory effects in different models. In a previous study on non-small cell lung cancer (NSCLC) tissues, we found that IDO2 expression revealed at the plasma membrane level of tumor cells was significantly associated with poor prognosis. In this study, the A549 human cell line, basally expressing IDO2, was used as an in vitro model of human lung adenocarcinoma to gain more insights into a possible alternative function of IDO2 different from the catalytic one. In these cells, immunocytochemistry and isopycnic sucrose gradient analyses confirmed the IDO2 protein localization in the cell membrane compartment, and the immunoprecipitation of tyrosine-phosphorylated proteins revealed that kinase activities can target IDO2. The different localization from the cytosolic one and the phosphorylation state are the first indications for the signaling function of IDO2, suggesting that the IDO2 non-enzymatic role in cancer cells is worthy of deeper understanding.

Indoleamine 2,3-dioxygenase 1 (IDO1) is a moonlight protein endowed with catalytic and signaling functions. It plays a pivotal role in the immune breaking mechanism leading to immunosuppression in cancer microenvironment. Intense research efforts have been devoted to designing catalytic inhibitors for developing immunotherapies, yet neglecting the enzyme’s signaling function. A few IDO1 inhibitors have reached the clinical stage, including navoximod, epacadostat and linrodostat. Using second harmonic generation analysis (SHG) and molecular dynamics simulations, here we show that these clinical inhibitors can induce distinct allosteric motions in the enzyme that affect the stability of the transient signaling complex between IDO1 and Src tyrosine kinase. Next generation sequencing demonstrates that, despite sharing a similar ability to inhibit the enzyme’s catalytic function, all three catalytic inhibitors modulate the IDO1’s signaling function in different ways, regulating distinct transcriptomes in SKOV3 cells.

Small synthetic oligodeoxynucleotides (ODNs) can mimic microbial nucleic acids by interacting with receptor systems and promoting immunostimulatory activities. Nevertheless, some ODNs can act differently on the plasmacytoid dendritic cell (pDC) subset, shaping their immunoregulatory properties and rendering them suitable immunotherapeutic tools in several clinical settings for treating overwhelming immune responses. We designed HIV–1–derived, DNA- and RNA-based oligonucleotides (gag, pol, and U5 regions) and assessed their activity in conferring a tolerogenic phenotype to pDCs in skin test experiments. RNA-but not DNA-oligonucleotides are capable of inducing tolerogenic features in pDCs. Interestingly, sensing the HIV–1–derived single-stranded RNA-gag oligonucleotide (RNA-gag) requires both TLR3 and TLR7 and the engagement of the TRIF adaptor molecule. Moreover, the induction of a suppressive phenotype in pDCs by RNA-gag is contingent upon the induction and activation of the immunosuppressive enzyme Arginase 1. Thus, our data suggest that sensing of the synthetic RNA-gag oligonucleotide in pDCs can induce a suppressive phenotype in pDCs, a property rendering RNA-gag a potential tool for therapeutic strategies in allergies and autoimmune diseases.

Indoleamine 2,3-dioxygenase 2 (IDO2) is a paralogue of IDO1, a tryptophan-degrading enzyme producing immunomodulatory molecules. However, the two proteins are unlikely to carry out the same functions. IDO2 shows little or no tryptophan catabolic activity and exerts contrasting immunomodulatory roles in a context-dependent manner, in both cancer and autoimmune diseases. The recently described potential non-enzymatic activity of IDO2 has suggested its possible involvement in alternative pathways resulting in either pro- or anti-inflammatory effects in different models. In a previous study on non-small cell lung cancer (NSCLC) tissues, we found that IDO2 expression, revealed at the plasma membrane level of tumor cells, was significantly associated with poor prognosis. In this study, the A549 human cell line, basally expressing IDO2, was used as an in vitro model of human lung adenocarcinoma to gain more insights on a possible alternative function of IDO2, different from the catalytic one. In these cells, immunocytochemistry and isopycnic sucrose gradient analyses confirmed the IDO2 protein localization in the cell membrane compartment, and immunoprecipitation of tyrosine-phosphorylated proteins revealed that IDO2 can be targeted by kinase activities. The different localization than the cytosolic one and the phosphorylation state are the first indications for the signaling function of IDO2, suggesting that the IDO2 non-enzymatic role in cancer cells is worthy of deeper understanding.

Immunoglobulin repertoires contain a fraction of antibodies that recognize low molecular weight compounds, including some enzymes’ cofactors, such as heme. Here, by using a set of 113 samples with variable region sequences matching clinical-stage antibodies, we demonstrated that a considerable number of these antibodies interact with heme. Antibodies that interact with heme possess specific sequence traits of their antigen-binding regions. Moreover they manifest particular physicochemical and functional qualities i.e. increased hydrophobicity, higher propensity of self-binding, higher intrinsic polyreactivity and reduced expression yields. Thus, interaction with heme is a strong predictor of different molecular and functional qualities of antibodies. Notably, these qualities are of high importance for therapeutic antibodies, as their presence was associated with failure of drug candidates to reach clinic. Our study reveled an important facet of information about relationship sequence-function in antibodies. It also offers a convenient tool for detection of liabilities of therapeutic antibodies.