A series of 2-(quinazolin-4-ylamino)-[1,4] benzoquinone derivatives that function as potent covalent-binding, irreversible inhibitors of the kinase domain of vascular endothelial growth factor receptor-2 (VEGFR-2) has been prepared by ceric ammonium nitrate oxidation of substituted (2,5-dimethoxyphenyl)(6,7-disubstituted-quinazolin-4-yl)amines and by displacement of the chlorine atom of substituted 2-chloro-5-(6,7-disubstituted-quinazolin-4-ylamino)-[1,4]benzoquinones with various amines, anilines, phenols, and alcohols. Enzyme studies were conducted in the absence and presence of glutathione and plasma. Several of the compounds inhibit VEGF-stimulated autophosphorylation in intact cells. Kinetic experiments were performed to study the reactivity of selected inhibitors toward glutathione. Reactivities correlated with LUMO energies calculated as averages of those of individual conformers weighted by the Boltzmann distribution. These results and molecular modeling were used to rationalize the biological observations. The compounds behave as non-ATP-competitive inhibitors. Unequivocal evidence, from mass spectral studies, indicates that these inhibitors form a covalent interaction with Cys-1045. One member of this series displays antitumor activity in an in vivo model.
Protein 4.2 (P4.2) is a major component of the erythrocyte plasma membrane accounting for approx. 5% of total membrane protein. The major membrane binding site for P4.2 is contained within the cytoplasmic domain of band 3 (cdb3), although the precise location of the cdb3 binding site is not known. To identify the cdb3 binding site, we used synthetic P4.2 peptides (15-mers) that spanned the entire 721-amino-acid large isoform of P4.2, and determined the binding of these peptides to cdb3 in an in vitro binding assay. One peptide, P8 (L61FVRRGQPFTIILYF), bound strongly to cdb3 and four others bound less strongly (P22, L271LNKRRGSVPILRQW; P27, G346EGQRGRIWIFQTST; P41, L556WRKKLHLTLSANLE; P48, I661HRERSYRFRSVWPE). These peptides have in common a cluster of two or three basic amino acid residues (arginine or lysine), in a region without nearby acidic residues. Cdb3 bound saturably to P8 with a Kd of 0.16 microM and a capacity of 0.56 mol of cdb3 monomer/mol of P8. Use of overlapping synthetic peptides further defined the cdb3 site as being contained within V63RRGQPFTIILYF. Replacement of R64R with R64G, G64R or G64G almost completely abolished cdb3 binding, suggesting that R64R is essential for cdb3 binding. P8 competitively inhibited binding of purified human erythrocyte P4.2 to cdb3. In blot overlay assays, cdb3 bound to a 23 kDa N-terminal P4.2 tryptic peptide containing V63RRGQPFTIILYF but not to other P4.2 tryptic peptides lacking this site. The V63RRGQPFTIILYF site is highly conserved in mouse and human erythrocyte P4.2 as well as between P4.2 and transglutaminase proteins, which are evolutionarily related to P4.2.
Abstract Antibody-drug conjugates (ADCs) enable targeted delivery of therapeutics to cancer cells and offer potential for more selective therapy. Several ADCs are demonstrating promising clinical efficacy, however due to the complexity of human cancer, tumors become refractory to most drug treatments. We hypothesized that cultured tumor cells chronically treated with an ADC would acquire mechanisms of resistance unique to ADC-based therapy. Human breast cancer cell lines were exposed to multiple cycles of an anti-Her2 trastuzumab-maytansinoid conjugate (TM) at IC80 concentrations for 3 days followed by ∼1 week without treatment to simulate a maximally tolerated dose followed by recovery. After ∼2 months, significant resistance developed in JIMT1 and MDA-MB-361 cell lines. The potency of TM conjugate on drug-selected cell lines was reduced to the activity observed on Her2-negative cells (>20 & >270X in JIMT1 & 361 cell models, respectively). Flow cytometry revealed 58% and 25% decreases in Her2 receptor number in JIMT-TM & 361-TM, respectively. Proteomic profiling of surface proteins in JIMT-TM cells demonstrated significant increases in proteins involved in post-translational modification (e.g., ubiquitinating enzymes, kinases, and phosphatases), as well as elevated levels of endosomal and vesicle proteins (e.g., RAB family members), and proteins mediating microtubule and actin dynamics. Notably, ABC drug transporters were not altered in JIMT-TM cells. In 361-TM cells, an increase in ABCC1 (MRP1) was observed, but no changes in ABCB1 (MDR1) which typically effluxes tubulin inhibitors. These data suggest the acquisition of complex resistance mechanisms upon ADC treatment. The cross-resistance profile of these ADC refractory models was evaluated. Minimal or no resistance (1 - 6X) was observed to free drugs, including maytansine or other standard-of-care tubulin or DNA targeted therapeutics. In JIMT-TM cells, cross-resistance was observed to other trastuzumab-based ADCs, including those containing either non-cleavable or cleavable linkers, and delivering payloads with various biological mechanisms of action. In contrast, 361-TM cells (which were made resistant to an ADC delivering a tubulin inhibitor via a non-cleavable linker) retained significant sensitivity to ADCs containing cleavable linkers, even those containing other tubulin inhibitor-based payloads. Moreover, both 361-TM and JIMT-TM resistant cell lines retained sensitivity to ADCs delivering payloads with alternative (non-tubulin) mechanisms of action. Therefore, despite the reduction in antigen levels observed in both cell lines, modification of the linker and/or the payload was able to overcome resistance mediated by the initial ADC therapy. These data offer the potential to treat refractory tumors with ADCs containing the same antibody vehicle, but delivering alternative linkers or payloads. Citation Format: Xingzhi Tan, Guixian Jin, Jeremy Myers, Veronica Diesl, Max Follettie, My-Hanh Lam, Sylvia Musto, Kiran Khandke, Manoj Charati, Edmund Graziani, Andreas Maderna, Chakrapani Subramanyam, Frank Koehn, Russell Dushin, Kim Arndt, Christopher J. O'Donnell, Hans-Peter Gerber, Frank Loganzo. Tumor cells selected for resistance to an antibody-drug conjugate retain sensitivity to ADCs with modified linkers and payloads. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4629. doi:10.1158/1538-7445.AM2013-4629
An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.
The focus of the antibody-drug conjugate (ADC) field is shifting toward development of site-specific, next-generation ADCs to address the issue of heterogeneity, metabolic instability, conjugatability, and less than ideal therapeutic index associated with the conventional (heterogeneous) ADCs. It is evident from the recent literature that the site of conjugation, the structure of the linker, and the physicochemical properties of the linker-payload all have a significant impact on the safety and efficacy of the resulting ADCs. Screening multiple linker-payloads on multiple sites of an antibody presents a combinatorial problem that necessitates high-throughput conjugation and purification methodology to identify ADCs with the best combination of site and payload. Toward this end, we developed a protein A/L-based solid-phase, site-specific conjugation and purification method that can be used to generate site-specific ADCs in a 96-well plate format. This solid-phase method has been shown to be versatile because of its compatibility with various conjugation functional handles such as maleimides, haloacetamides, copper free click substrates, and transglutaminase substrates. The application of this methodology was further expanded to generate dual labeled, site-specific antibody and Fab conjugates.
Abstract Antibody drug conjugates (ADCs) are designed to deliver cytotoxics to tumor cells via binding to surface antigen followed by internalization and intracellular drug release. ADC linkers are typically categorized as non-cleavable or cleavable; a cleavable linker example is Y_mcValCitPABC_X, with antibody Y, a dipeptide sequence with self-immolative PABC spacer, and payload X. This linker is known to be cleaved by endosomal/lysosomal proteases such as cathepsins, releasing attached payload. In addition to intracellular processing of this linker, we report that conditioned media of cultured tumor cell lines is sufficient to promote extracellular cleavage of ADCs with peptide-linked payloads. Cultured cell lines N87 (gastric) and U87 (glioblastoma), and patient-derived xenograft PA0165 (pancreatic) adapted to in vitro culture, were plated either in standard 2D culture or in 3D Cultrex embedded culture. After 3 - 7 days, conditioned media from cells was transferred onto MDA-MB-468 or HT29 cells, and then ADCs (Y_mcValCitPABC_Aur) were added to cultures. ADCs were non-targeting IgG conjugated via cleavable dipeptide-PABC linker to auristatin tubulin inhibitor. Minimal cytotoxicity was observed with ADC alone on 468 or HT29 cells. However, in the presence of conditioned media from N87, U87, or PA0165 cells plus the ADC, cytotoxicity was observed in the recipient cells (up to 31, 22, 56% growth inhibition respectively at 100 nM ADC). Moreover, in all cases, the magnitude of the response was greatest when cells providing conditioned media were grown in 3D culture (up to 56, 48, 70%, respectively). In contrast, minimal response was observed using conditioned media from other cancer cell lines (ie HCC2429, 1 - 17%). Additional analyses were conducted by incubating conditioned media from these cells with a dipeptide-based cleavable substrate with fluorescent probe and measuring released product in a plate-based assay. Conditioned media promoted fluorescence, suggesting proteolytic enzymes secreted by cells. An ELISA confirmed the presence of cathepsins in conditioned media. Complementing these studies, proteolytic activity was detected in the interstitial fluid derived from tumors grown in athymic mice. Fluid extracted from xenograft tumors (cultured cancer lines and patient-derived tumors) was analyzed for proteolytic activity using cleavable-fluorescent linker-probe in a plate assay. The majority of samples demonstrated proteolytic activity. These data are consistent with reported secretion of cathepsins by cancer cells and we now show that these proteases may mediate extracellular release of cytotoxic payloads from ADCs containing peptide-based cleavable linkers. This activity is magnified when cells are grown in 3D culture and is observed in tumor xenografts grown in vivo. This response may provide a beneficial bystander effect of ADCs on antigen negative cells in a heterogenous tumor population. Citation Format: My-Hanh Lam, Judy Lucas, Andreas Maderna, Hallie Wald, Megan Wojciechowicz, Russell Dushin, Bryan Peano, Fang Wang, Jeremy Myers, Xingzhi Tan, Sylvia Musto, Manoj Charati, Hans-Peter Gerber, Frank Loganzo. Extracellular proteolytic cleavage of peptide-linked antibody-drug conjugates promotes bystander killing of cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4837. doi:10.1158/1538-7445.AM2014-4837
A260 Plk1 is a Ser/Thr kinase that regulates numerous events during mitosis. Plk1 is over-expressed in human tumors and its inhibition by siRNA knock-down and other methods has validated the potential utility of Plk1 kinase inhibitors in cancer. Assays have previously been developed to evaluate Plk1 inhibitors using casein, a generic kinase substrate. However, the need to evaluate kinase activity in cells by monitoring an immediate downstream substrate specific for the kinase is critical for inhibitor development. Plk1 was previously reported to phosphorylate Myt1 at T495, which attenuates Myt1 activity and releases inhibition of Cdk1/cyclin B. A polyclonal antibody to a peptide containing phospho-T495-Myt1 was generated. A 384-well, non-radioactive DELFIA kinase assay was developed using bacterially expressed Plk1 protein, biotinylated-Myt1 peptide, and anti-P-T495-Myt1 antibody. High signal-to-noise ratios (>100X) were achieved using low concentrations of Plk1. A homogenous LANCE assay was also developed to enable HTS. Anti-P-T495-Myt1 antibody and pre-activation of Plk1 with ATP were critical for LANCE assay development since a generic anti-P-Thr antibody and unactivated Plk1 could not achieve adequate signals. Benchmark inhibitors produced equivalent IC50’s in both assay formats. P-T495-Myt1 was explored as a potential biomarker of Plk1 kinase activity in cells. Plk1 protein levels or kinase activity were diminished in cells by siRNA or small molecule kinase inhibitors, respectively. Decreases in P-T495-Myt1 signals were observed in treated cells by immunoblot analyses without decreases in total Myt1 protein levels. These data indicate that Plk1 kinase activity can be monitored in cells with anti-P-T495-Myt1 antibody. An immune-complex kinase assay was also developed to further evaluate Plk1 kinase activity in cells. Kinase reactions containing GST-Myt1, ATP, and Plk1 immunoprecipitated from mitotic cells were fractionated and evaluated for P-T495-Myt1 by immunoblot. Benchmark inhibitors produced dose-dependent decreases of P-Myt1 signal, consistent with loss of Plk1 kinase activity. These data support the use of P-T495-Myt1 antibody as a tool to evaluate the activity of Plk1 kinase inhibitors in plate-based primary screening assays and as a biomarker of Plk1 kinase activity in cells.
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