Visualizing drug-induced lipid accumulation in lysosomes of live cancer cells with stimulated Raman imaging

The low pH of the lysosomal compartment often results in sequestration of chemotherapeutic agents that contain positively charged basic functional groups, leading to anti-cancer drug resistance. To visualize drug localization in lysosomes and its influence on lysosomal functions, we synthesize a group of drug-like compounds that contain both a basic functional group and a bisarylbutadiyne (BADY) group as a Raman probe. With quantitative stimulated Raman scattering (SRS) imaging, we validate that the synthesized lysosomotropic (LT) drug analogs show high lysosomal affinity, which can also serve as a photostable lysosome tracker. We find that long-term retention of the LT compounds in lysosomes leads to the increased amount and colocalization of both lipid droplets (LDs) and lysosomes in SKOV3 cells. With hyperspectral SRS imaging, further studies find that the LDs stuck in lysosomes are more saturated than the LDs staying out of the lysosomes, indicating impaired lysosomal lipid metabolism by the LT compounds. These results demonstrate that SRS imaging of the alkyne-based probes is a promising approach to characterizing the lysosomal sequestration of drugs and its influence on cell functions

Esters in ADC linkers: Optimization of stability and lysosomal cleavage

Antibody-drug conjugates (ADCs) present a unique opportunity to increase the safety of highly toxic drugs by utilizing the specificity of antibodies to deliver (typically amine-containing) payloads to specific tissues. However, there are few technologies for attaching alcohol-containing payloads to an antibody. Theoretically, this would be simplest with the use of an ester linkage. However, little is known about the lysosomal and plasma stability of ester linkages on ADCs. Herein, we describe various experiments evaluating this stability in both human/mouse plasma and in lysosomes, as well as the design of a lysosomally cleavable peptide-linked ester. The stability of ester-linked payloads was demonstrated by observing an ester-linked ADC in the presence of lysosomes. The ester remained intact while the antibody backbone was proteolytically degraded. The lysosomal stability of this linkage was additionally confirmed in a cell-based system using SKBR3 cells. We next undertook the design of a novel linker that is stable in mouse plasma, but upon lysosomal uptake, undergoes immolation, resulting in spontaneous ester cleavage. With this research, we hope to further our knowledge of ester linkages in ADCs as well as to develop a feasible way to release an unmodified alcohol-containing drug into a chosen cell type.https://orb.binghamton.edu/research_days_posters_spring2020/1093/thumbnail.jp

Indoloquinoline-Mediated Targeted Downregulation of KRAS through Selective Stabilization of the Mid-Promoter G-Quadruplex Structure

KRAS is a well-validated anti-cancer therapeutic target, whose transcriptional downregulation has been demonstrated to be lethal to tumor cells with aberrant KRAS signaling. G-quadruplexes (G4s) are non-canonical nucleic acid structures that mediate central dogmatic events, such as DNA repair, telomere elongation, transcription and splicing events. G4s are attractive drug targets, as they are more globular than B-DNA, enabling more selective gene interactions. Moreover, their genomic prevalence is increased in oncogenic promoters, their formation is increased in human cancers, and they can be modulated with small molecules or targeted nucleic acids. The putative formation of multiple G4s has been described in the literature, but compounds with selectivity among these structures have not yet been able to distinguish between the biological contribution of the predominant structures. Using cell free screening techniques, synthesis of novel indoloquinoline compounds and cellular models of KRAS-dependent cancer cells, we describe compounds that choose between KRAS promoter G4near and G4mid, correlate compound cytotoxic activity with KRAS regulation, and highlight G4mid as the lead molecular non-canonical structure for further targeting efforts

Development of Solid-Phase Site-Specific Conjugation and Its Application toward Generation of Dual Labeled Antibody and Fab Drug Conjugates

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

Molecular Validation of LpxC as an Antibacterial Drug Target in Pseudomonas aeruginosa

LpxC [UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc deacetylase] is a metalloamidase that catalyzes the first committed step in the biosynthesis of the lipid A component of lipopolysaccharide. A previous study (H. R. Onishi, B. A. Pelak, L. S. Gerckens, L. L. Silver, F. M. Kahan, M. H. Chen, A. A. Patchett, S. M. Galloway, S. A. Hyland, M. S. Anderson, and C. R. H. Raetz, Science 274:980-982, 1996) identified a series of synthetic LpxC-inhibitory molecules that were bactericidal for Escherichia coli. These molecules did not inhibit the growth of Pseudomonas aeruginosa and were therefore not developed further as antibacterial drugs. The inactivity of the LpxC inhibitors for P. aeruginosa raised the possibility that LpxC activity might not be essential for all gram-negative bacteria. By placing the lpxC gene of P. aeruginosa under tight control of an arabinose-inducible promoter, we demonstrated the essentiality of LpxC activity for P. aeruginosa. It was found that compound L-161,240, the most potent inhibitor from the previous study, was active against a P. aeruginosa construct in which the endogenous lpxC gene was inactivated and in which LpxC activity was supplied by the lpxC gene from E. coli. Conversely, an E. coli construct in which growth was dependent on the P. aeruginosa lpxC gene was resistant to the compound. The differential activities of L-161,240 against the two bacterial species are thus the result primarily of greater potency toward the E. coli enzyme rather than of differences in the intrinsic resistance of the bacteria toward antibacterial compounds due to permeability or efflux. These data validate P. aeruginosa LpxC as a target for novel antibiotic drugs and should help direct the design of inhibitors against clinically important gram-negative bacteria

Multivalent peptidic linker enables identification of preferred sites of conjugation for a potent thialanstatin antibody drug conjugate.

Antibody drug conjugates (ADCs) are no longer an unknown entity in the field of cancer therapy with the success of marketed ADCs like ADCETRIS and KADCYLA and numerous others advancing through clinical trials. The pursuit of novel cytotoxic payloads beyond the mictotubule inhibitors and DNA damaging agents has led us to the recent discovery of an mRNA splicing inhibitor, thailanstatin, as a potent ADC payload. In our previous work, we observed that the potency of this payload was uniquely tied to the method of conjugation, with lysine conjugates showing much superior potency as compared to cysteine conjugates. However, the ADC field is rapidly shifting towards site-specific ADCs due to their advantages in manufacturability, characterization and safety. In this work we report the identification of a highly efficacious site-specific thailanstatin ADC. The site of conjugation played a critical role on both the in vitro and in vivo potency of these ADCs. During the course of this study, we developed a novel methodology of loading a single site with multiple payloads using an in situ generated multi-drug carrying peptidic linker that allowed us to rapidly screen for optimal conjugation sites. Using this methodology, we were able to identify a double-cysteine mutant ADC delivering four-loaded thailanstatin that was very efficacious in a gastric cancer xenograft model at 3mg/kg and was also shown to be efficacious against T-DM1 resistant and MDR1 overexpressing tumor cell lines