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Huntington's disease neurodegeneration occurs when the number of consecutive glutamines in the huntingtin exon-1 (HTTExon1) exceeds a pathological threshold of 35. The sequence homogeneity of HTTExon1 reduces the signal dispersion in NMR spectra, hampering its structural characterization. By simultaneously introducing three isotopically labeled glutamines in a site-specific manner in multiple concatenated samples, 18 glutamines of a pathogenic HTTExon1 with 36 glutamines were unambiguously assigned. Chemical shift analyses indicate the α-helical persistence in the homorepeat and the absence of an emerging toxic conformation around the pathological threshold. Using the same type of samples, the recognition mechanism of Hsc70 molecular chaperone has been investigated, indicating that it binds to the N17 region of HTTExon1, inducing the partial unfolding of the poly-Q. The proposed strategy facilitates high-resolution structural and functional studies in low-complexity regions., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Huntington's disease is a neurodegenerative disorder caused by a CAG expansion in the first exon of the HTT gene, resulting in an extended polyglutamine (poly-Q) tract in huntingtin (httex1). The structural changes occurring to the poly-Q when increasing its length remain poorly understood due to its intrinsic flexibility and the strong compositional bias. The systematic application of site-specific isotopic labeling has enabled residue-specific NMR investigations of the poly-Q tract of pathogenic httex1 variants with 46 and 66 consecutive glutamines. Integrative data analysis reveals that the poly-Q tract adopts long α-helical conformations propagated and stabilized by glutamine side chain to backbone hydrogen bonds. We show that α-helical stability is a stronger signature in defining aggregation kinetics and the structure of the resulting fibrils than the number of glutamines. Our observations provide a structural perspective of the pathogenicity of expanded httex1 and pave the way to a deeper understanding of poly-Q-related diseases., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Multidrug resistance (MDR) is a major challenge in cancer treatment, and the breast cancer resistance protein (BCRP) is an important target in the search for new MDR-reversing drugs. With the aim of discovering new potential BCRP inhibitors, the crude extract of leaves of Eremophila galeata , a plant endemic to Australia, was investigated for inhibitory activity of parental (HT29 par ) as well as BCRP-overexpressing HT29 colon cancer cells resistant to the chemotherapeutic SN-38 (i.e., HT29 SN38 cells). This identified a fraction, eluted with 40% acetonitrile on a solid-phase extraction column, which showed weak growth-inhibitory activity on HT29 SN38 cells when administered alone, but exhibited concentration-dependent growth inhibition when administered in combination with SN-38. The major constituent in this fraction was isolated and found to be 5,3',5'-trihydroxy-3,6,7,4'-tetramethoxyflavone ( 2 ), which at a concentration of 25 μg/mL potentiated the growth-inhibitory activity of SN-38 to a degree comparable to that of the known BCRP inhibitor Ko143 at 1 μM. A dye accumulation experiment suggested that 2 inhibits BCRP, and docking studies showed that 2 binds to the same BCRP site as SN-38. These results indicate that 2 acts synergistically with SN-38, with 2 being a BCRP efflux pump inhibitor while SN-38 inhibits topoisomerase-1.

ATP-binding cassette (ABC) transporters, such as breast cancer resistance protein (BCRP), are key players in resistance to multiple anti-cancer drugs, leading to cancer treatment failure and cancer-related death. Currently, there are no clinically approved drugs for reversal of cancer drug resistance caused by ABC transporters. This study investigated if a novel drug candidate, SCO-201, could inhibit BCRP and reverse BCRP-mediated drug resistance. We applied in vitro cell viability assays in SN-38 (7-Ethyl-10-hydroxycamptothecin)-resistant colon cancer cells and in non-cancer cells with ectopic expression of BCRP. SCO-201 reversed resistance to SN-38 (active metabolite of irinotecan) in both model systems. Dye efflux assays, bidirectional transport assays, and ATPase assays demonstrated that SCO-201 inhibits BCRP. In silico interaction analyses supported the ATPase assay data and suggest that SCO-201 competes with SN-38 for the BCRP drug-binding site. To analyze for inhibition of other transporters or cytochrome P450 (CYP) enzymes, we performed enzyme and transporter assays by in vitro drug metabolism and pharmacokinetics studies, which demonstrated that SCO-201 selectively inhibited BCRP and neither inhibited nor induced CYPs. We conclude that SCO-201 is a specific, potent, and potentially non-toxic drug candidate for the reversal of BCRP-mediated resistance in cancer cells., Competing Interests: Steffen Rump (S.R.) and Henning Weigt (H.W.) hold patent application PCT/EP2016/053843 on pyrazolo-pyrimidine derivatives as BCRP inhibitors. Jan Stenvang (J.S.) and Nils Brünner (N.B.) are co-founders of Scandion Oncology A/S who own all rights to SCO-201. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.