Indirubins: A Potential Therapeutic Target in Multiple Myeloma
Authors: Bagratuni Tina , Gaboriad-Kolar Nicolas , Zakopoulou Roubini , Myrianthopoulos Vassilios , Kastritis Efstathios , Terpos Evangelos , Mikros Emmanuel , Skaltsounis Alexios-Leandros , Dimopoulos Meletios A 
Published: October 2019

Abstract
Current drugs in the treatment of Multiple Myeloma (MM) result in cell death via a number of mechanisms including a direct effect on plasma cells as well as alteration in the BM microenvironment. Although effective to some extent, none of the drug mechanisms of action are fully targeting a biological process essential and necessary for PC survival. Among the FDA approved kinase inhibitors, few are based on natural scaffolds. 6-bromoindirubin-3'-oxime (6BIO) is a potent kinase inhibitor based on the natural 6-bromoindirubin scaffold. Indirubin and 6-bromoindirubin are two natural products that have found a particular interest in dye chemistry as the main constituent of indigo and Tyrian purple dyes. Recent findings discovered that 6BIO was a promising anti-cancer agent acting on the JAK/STAT signaling pathway mediating cell proliferation. After enhancement of the chemical structure of 6BIO, further reports exposed that MLS-2438 and MLS-2384 were Akt signaling pathway inhibitor (MLS-2438) and potent c-Src kinase direct inhibitors.
A library containing 2000 natural molecules was constructed using several data platforms. Each molecule was processed through different filters such as tautomeric studies, protonation and steroisomerism status in order to be used for calculations of virtual evaluation. Two approaches were followed: the structure-based virtual screening and the ligand-based virtual screening. To achieve structural based virtual screening, binding and evaluation of the chemical relation of each molecule in the crystallographic structure of the proteasome β5 subunit was performed. In the ligand-based virtual screening, calculations were made to identify the structural similarities of each molecule with the known proteasome inhibitor, bortezomib.
The results of both approaches were combined, the molecules ranked, and 100 out of 2000 were identified as strong potential bioactive hits for the β5 subunit. Out of these 100 molecules, the chemical structures of high interest were the following: indole alkaloids derivatives (indirubins), flavonoids, secoiridoids, simple phenolic acids and acetophenone. A rational selection of indirubins derivatives was conducted in order to study their cytotoxic effects on MM. Fifty indirubins derivatives were selected based on different criteria: structure, known/unknown targets, chemodiversity in substitution patterns.
To explore the inhibitory effects of indirubins in MM, we performed the WST1 proliferation assay in three MM cell lines (H929, JJN3, L363). Initially, all the selected indirubins (~50 indirubins) were tested at 7.5μM in L363 cell line and proliferation results from the WST1 assay extracted after 24 hours of treatment. More than half of the indirubins tested displayed more than 50% reduction of the proliferation at 7.5μM. Interestingly, 10 out of the 50 indirubins tested reduced more than 80% proliferation after 24 hours. The most active indirubins were tested in H929 and JJN3 cell lines, where similar effects were seen after 24 hours of treatment. All tested indirubins acted in a dose-dependent manner. Based on our first set of data, we suggest that indirubins have significant anti-proliferative effects on MM cell lines.
Among the most active indirubins, two molecules namely 805 and 673 emerged as attractive for further development. Compound 805 is an analog of MLS-2384 while compound 673 is an analog of MLS-2438. The latter derivative represents a promising candidate displaying an IC50below the micromolar range on H929 and JJN3 cells. To determine the kind of cell death caused by one of the most active indirubins, 673, cell cycle analysis was performed before and after treatment in H929 cell line. In particular changes in RNA expression of 84 genes key to cell cycle regulation were analyzed in H929 cell line. Our results show that among other genes, the ones which have a dramatic increase in their expression (>5 fold) are mainly involved in cell cycle arrest such as GADD45A, CDC34, TP53, CHEK1 and CHEK2. A more detailed analysis of the profiler array will be presented at the meeting.
In conclusion, this is the first study to show the inhibitory effects of indirubins in MM. Further investigation of these compounds may offer a therapeutic advantage that would affect MM pathogenesis and treatment.

Disclosures
Kastritis: Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Genesis: Consultancy, Honoraria. Terpos:Amgen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. Dimopoulos:Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Genesis: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.