Microcystins Containing Doubly Homologated Tyrosine Residues from a <i>Microcystis aeruginosa</i> Bloom: Structures and Cytotoxicity

Four new microcystin congeners are described including the first three examples of microcystins containing the rare doubly homologated tyrosine residue 2-amino-5-(4-hydroxyphenyl)­pentanoic acid (Ahppa) (<b>1</b>–<b>4</b>). Large-scale harvesting and biomass processing allowed the isolation of substantial quantities of these compounds, thus enabling complete structure determination by NMR as well as cytotoxicity evaluation against selected cancer cell lines. The new Ahppa-toxins all incorporate Ahppa residues at the 2-position, and one of these also has a second Ahppa at position 4. The two most lipophilic Ahppa-containing microcystins showed 10-fold greater cytotoxic potency against human tumor cell lines (A549 and HCT-116) compared to microcystin-LR (<b>5</b>). The presence of an Ahppa residue in microcystin congeners is difficult to ascertain by MS methods alone, due to the lack of characteristic fragment ions derived from the doubly homologated side chain. Owing to their unexpected cytotoxic potency, the potential impact of the compounds on human health should be further evaluated

Generation of site-specific multiple-payload carrying peptidic linker (MPP) ADC delivering MMAD.

<p>(A) Schematic showing generation of a double-MMAD carrying peptidic linker ADC generated on trastuzumab A114C. (B) <i>In vitro</i> cytotoxicity of peptidic linked MMAD trastuzumab A114C ADC against various levels of Her2 expressing cancer cell lines, reported in Mean IC₅₀ values of conjugated payload in nM. Data are the mean of multiple experiments. MAL = malemide; DBCO = Dibenzocyclooctyne.</p

In vitro cytotoxicity of single-cysteine mutant thailanstatin trastuzumab ADCs against various levels of Her2 expressing cancer cell lines, reported in Mean IC₅₀ values of conjugated payload in nM.

<p>In vitro cytotoxicity of single-cysteine mutant thailanstatin trastuzumab ADCs against various levels of Her2 expressing cancer cell lines, reported in Mean IC₅₀ values of conjugated payload in nM.</p

Potency of hinge-cysteine thailanstatin trastuzumab ADC.

<p>(A) Structure of iodoacetamide derivatized non-cleavable thailanstatin linker-payloads (LPs). (B) <i>In vitro</i> cytotoxicity of hinge-cysteine thailanstatin trastuzumab ADCs against cancer cell lines expressing various levels of Her2, reported in half-maximal inhibitory concentration (IC₅₀) values of conjugated payload in nM. Data are the mean of multiple experiments. (C) <i>In vivo</i> efficacy of hinge-cysteine thailanstatin trastuzumab <b>ADC1</b> in an N87 gastric cancer xenograft model dosed at 3 mg/kg (q4d x 4). Arrows indicate the day(s) on which intravenous dosing was carried out. DAR = Drug Antibody Ratio; 361 = MDA-MB-361-DYT2; 468 = MDA-MB-468.</p

Double-cysteine mutant thailanstatin trastuzumab ADCs.

<p>(A) <i>In vitro</i> cytotoxicity of double-cysteine mutant thailanstatin trastuzumab ADCs against various levels of Her2 expressing cancer cell lines, reported in Mean IC₅₀ values of conjugated payload in nM. Data are the mean of multiple experiments. (B) <i>In vivo</i> efficacy of double-cysteine mutant thailanstatin trastuzumab <b>ADC16</b> in N87 gastric cancer xenograft model dosed at 0.5, 1.56 and 3 mg/kg (q4d x 4). (C) <i>In vitro</i> cytotoxicity of double-cysteine mutant thailanstatin trastuzumab <b>ADC16</b> against T-DM1 resistant N87 (N87-TDM1) and 361 (361-TDM1) as well as MDR1 overexpressing N87 (N87-MDR1-CL3) cancer cell lines, reported in IC₅₀ values of conjugated payload in nM.</p

Natural Product Splicing Inhibitors: A New Class of Antibody–Drug Conjugate (ADC) Payloads

There is a considerable ongoing work to identify new cytotoxic payloads that are appropriate for antibody-based delivery, acting via mechanisms beyond DNA damage and microtubule disruption, highlighting their importance to the field of cancer therapeutics. New modes of action will allow a more diverse set of tumor types to be targeted and will allow for possible mechanisms to evade the drug resistance that will invariably develop to existing payloads. Spliceosome inhibitors are known to be potent antiproliferative agents capable of targeting both actively dividing and quiescent cells. A series of thailanstatin–antibody conjugates were prepared in order to evaluate their potential utility in the treatment of cancer. After exploring a variety of linkers, we found that the most potent antibody–drug conjugates (ADCs) were derived from direct conjugation of the carboxylic acid-containing payload to surface lysines of the antibody (a “linker-less” conjugate). Activity of these lysine conjugates was correlated to drug-loading, a feature not typically observed for other payload classes. The thailanstatin-conjugates were potent in high target expressing cells, including multidrug-resistant lines, and inactive in nontarget expressing cells. Moreover, these ADCs were shown to promote altered splicing products in N87 cells in vitro, consistent with their putative mechanism of action. In addition, the exposure of the ADCs was sufficient to result in excellent potency in a gastric cancer xenograft model at doses as low as 1.5 mg/kg that was superior to the clinically approved ADC T-DM1. The results presented herein therefore open the door to further exploring splicing inhibition as a potential new mode-of-action for novel ADCs