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Niche-Based Screening in Multiple Myeloma Identifies a Kinesin-5 Inhibitor with Improved Selectivity over Hematopoietic Progenitors.

ABSTRACT: Novel therapeutic approaches are urgently required for multiple myeloma (MM). We used a phenotypic screening approach using co-cultures of MM cells with bone marrow stromal cells to identify compounds that overcome stromal resistance. One such compound, BRD9876, displayed selectivity over normal hematopoietic progenitors and was discovered to be an unusual ATP non-competitive kinesin-5 (Eg5) inhibitor. A novel mutation caused resistance, suggesting a binding site distinct from known Eg5 inhibitors, and BRD9876 inhibited only microtubule-bound Eg5. Eg5 phosphorylation, which increases microtubule binding, uniquely enhanced BRD9876 activity. MM cells have greater phosphorylated Eg5 than hematopoietic cells, consistent with increased vulnerability specifically to BRD9876's mode of action. Thus, differences in Eg5-microtubule binding between malignant and normal blood cells may be exploited to treat multiple myeloma. Additional steps are required for further therapeutic development, but our results indicate that unbiased chemical biology approaches can identify therapeutic strategies unanticipated by prior knowledge of protein targets.

SUBMITTER: Chattopadhyay S

PROVIDER: S-EPMC4524791 | biostudies-literature | 2015 Feb

REPOSITORIES: biostudies-literature

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Niche-Based Screening in Multiple Myeloma Identifies a Kinesin-5 Inhibitor with Improved Selectivity over Hematopoietic Progenitors.

Chattopadhyay Shrikanta S Stewart Alison L AL Mukherjee Siddhartha S Huang Cherrie C Hartwell Kimberly A KA Miller Peter G PG Subramanian Radhika R Carmody Leigh C LC Yusuf Rushdia Z RZ Sykes David B DB Paulk Joshiawa J Vetere Amedeo A Vallet Sonia S Santo Loredana L Cirstea Diana D DD Hideshima Teru T Dančík Vlado V Majireck Max M MM Hussain Mahmud M MM Singh Shambhavi S Quiroz Ryan R Iaconelli Jonathan J Karmacharya Rakesh R Tolliday Nicola J NJ Clemons Paul A PA Moore Malcolm A S MAS Stern Andrew M AM Shamji Alykhan F AF Ebert Benjamin L BL Golub Todd R TR Raje Noopur S NS Scadden David T DT Schreiber Stuart L SL

Cell reports 20150205 5

Novel therapeutic approaches are urgently required for multiple myeloma (MM). We used a phenotypic screening approach using co-cultures of MM cells with bone marrow stromal cells to identify compounds that overcome stromal resistance. One such compound, BRD9876, displayed selectivity over normal hematopoietic progenitors and was discovered to be an unusual ATP non-competitive kinesin-5 (Eg5) inhibitor. A novel mutation caused resistance, suggesting a binding site distinct from known Eg5 inhibito ...[more]

PMID: 25660025

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Project description:Parvovirus B19 (B19V) infection can cause hematological disorders and fetal hydrops during pregnancy. Currently, no antivirals or vaccines are available for the treatment or prevention of B19V infection. To identify novel small-molecule antivirals against B19V replication, we developed a high-throughput screening (HTS) assay, which is based on an in vitro nicking assay using recombinant N-terminal amino acids 1 to 176 of the viral large nonstructural protein (NS1N) and a fluorescently labeled DNA probe (OriQ) that spans the nicking site of the viral DNA replication origin. We collectively screened 17,040 compounds and identified 2,178 (12.78%) hits that possess >10% inhibition of the NS1 nicking activity, among which 84 hits were confirmed to inhibit nicking in a dose-dependent manner. Using ex vivo-expanded primary human erythroid progenitor cells (EPCs) infected by B19V, we validated 24 compounds that demonstrated >50% in vivo inhibition of B19V infection at 10 μM, which can be categorized into 7 structure scaffolds. Based on the therapeutic index (half-maximal cytotoxic concentration [CC50]/half-maximal effective concentration [EC50] ratio) in EPCs, the top 4 compounds were chosen to examine their inhibitions of B19V infection in EPCs at two times of the 90% maximal effective concentration (EC90). A purine derivative (P7) demonstrated an antiviral effect (EC50 = 1.46 μM) without prominent cytotoxicity (CC50 = 71.8 μM) in EPCs and exhibited 92% inhibition of B19V infection in EPCs at 3.32 μM, which can be used as the lead compound in future studies for the treatment of B19V infection-caused hematological disorders. IMPORTANCE B19V encodes a large nonstructural protein, NS1. Its N-terminal domain (NS1N) consisting of amino acids 1 to 176 binds to viral DNA and serves as an endonuclease to nick the viral DNA replication origins, which is a pivotal step in rolling-hairpin-dependent B19V DNA replication. For high-throughput screening (HTS) of anti-B19V antivirals, we miniaturized a fluorescence-based in vitro nicking assay, which employs a fluorophore-labeled probe spanning the terminal resolution site (trs) and the NS1N protein, into a 384-well-plate format. The HTS assay showed high reliability and capability in screening 17,040 compounds. Based on the therapeutic index (half-maximal cytotoxic concentration [CC50]/half-maximal effective concentration [EC50] ratio) in EPCs, a purine derivative demonstrated an antiviral effect of 92% inhibition of B19V infection in EPCs at 3.32 μM (two times the EC90). Our study demonstrated a robust HTS assay for screening antivirals against B19V infection.

Dataset Information

Niche-Based Screening in Multiple Myeloma Identifies a Kinesin-5 Inhibitor with Improved Selectivity over Hematopoietic Progenitors.

Publications

Niche-Based Screening in Multiple Myeloma Identifies a Kinesin-5 Inhibitor with Improved Selectivity over Hematopoietic Progenitors.

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