Biology subjects

Mistry, R.

Publications and source records attributed to Mistry, R..

6 recordsLinked to original sources

bioRxiv2026

Genetically-encoded discovery and development of peptide-macrocycle imaging agents for PD-L1

The unique cell surface composition of tumor cells forms the molecular basis for many targeting and cell-based therapies. Here, we describe the development of novel peptide-based targeting agents for programmed death ligand 1 (PD-L1). Molecular imaging by peptide agents, coupled with therapeutic intervention using the same modality, represents a critical advancement in cancer management. Whole-body PET imaging of PD-L1 expression offers a superior alternative to traditional immuno-histochemistry, making PD-L1 radiodiagnostic imaging a highly sought-after modality. PD-L1 targeting modalities developed for clinical imaging to date can be divided into antibodies, protein domains, and small macrocyclic peptides with fewer than 20 amino acids. The latter modalities can address many challenges seen in antibody-based targeting vectors. All potent PD-L1 targeting peptide modalities reported to date rely extensively on non-canonical amino acids (ncAAs). Here, we report a comprehensive structure-activity relationship (SAR) analysis of a family of macrocycles discovered from an Sx2Cx8Cx2 phage-display library composed entirely of natural amino acids (x represents 19 natural amino acids excluding Cys). Using >10,000 variants in ''focused'' phage-display libraries, we optimized these macrocycles to achieve single-digit-nanomolar potency in protein- and cell-based assays. En route to this optimization, the activity of 216 synthetic macrocycles towards PD-L1 was measured in five distinct assays; two leads have been evaluated by imaging in tumor xenografts in mice, and the X-ray structure of one advanced lead in complex with PD-L1 has been determined at 2.78 [A] resolution. This publication demonstrates the development potential of PD-L1-targeting macrocycles that do not require extensive incorporation of ncAAs and the democratization of discovery by mapping the optimization path to single-digit-nanomolar assets for targeted radiopharmaceuticals via canonical phage-display technology.

pharmacology and toxicology↗

bioRxiv2026

DNA Damage Driven Viability Loss and Transcriptional Reprogramming in Chinese Hamster Ovary Cell Perfusion Culture

Intensified perfusion cultures promise higher yields and consistent product quality, yet extended runs frequently stall due to declining Chinese hamster ovary (CHO) cell viability at high cell density. Here, we identify DNA damage accumulation as a central, previously underappreciated driver of this limitation. Using two antibody-expressing CHO cell lines operated at a control and high cell density in perfusion bioreactors, we combined time-resolved transcriptomics with molecular, biophysical and super-resolution imaging analyses. We observed a progressive, global downregulation of DNA damage response (DDR) pathways accompanied by a time-dependent accumulation of DNA lesions. Notably, {gamma}H2AX signalling declined despite DNA damage, indicating impaired damage sensing and repair. Concomitantly, RNA polymerase II protein levels and transcriptional hub organisation were markedly reduced, consistent with widespread transcriptional dysfunction preceding loss of viability. An extended 21-day perfusion run confirmed continued viability decline beyond day 14, supporting a cumulative damage model. Comparison with HEK293 cells revealed substantially more efficient DNA repair, underscoring an intrinsically attenuated DDR in CHO cells. Together, these findings demonstrate that unrepaired DNA damage fundamentally constrains perfusion culture longevity, exposing limits of CHO genomic plasticity and highlighting DDR pathways as promising targets for host cell engineering to enhance perfusion performance.

bioengineering↗

bioRxiv2026

FLEX: A heparin-binding fusion partner engineered from fibroblast growth factor 1 to enhance protein expression, solubility and purity

Expression of aggregation-prone, unstable, or cytotoxic recombinant proteins remains a major bottleneck in both academic and industrial research. Although solubility-enhancing affinity tags can improve expression, they often compromise purification stringency, increase construct size, or require additional downstream processing. Here we report FLEX, a compact 15.5-kDa dual-function fusion tag engineered from human fibroblast growth factor-1 (FGF1) that integrates intrinsic protein-stabilising properties with high-affinity heparin binding. Structure-guided computational redesign of the FGF1 scaffold reduced exposed hydrophobic residues, removed flexible protease-susceptible regions, and expanded the electropositive surface while preserving the canonical heparin-binding interface. FLEX exhibits markedly improved thermal and chemical stability relative to wild-type FGF1 together with enhanced heparin affinity, enabling high-stringency washing and improved purity in a single affinity step. We demonstrate the broad utility of FLEX by expressing and purifying a panel of challenging proteins in Escherichia coli, including cytotoxic Pseudomonas aeruginosa virulence factors that are difficult to obtain in active form. Unexpectedly, FLEX also performed robustly in mammalian expression systems, where transiently expressed FLEX-tagged proteins were recovered at higher yield and purity than with gold standard Myc and Strep tags, including difficult targets such as Tribbles 3 (TRIB3). These findings establish FLEX as a versatile affinity-and-stabilisation tag that improves expression and purification across diverse systems, providing a practical new tool for structural, biochemical, and translational studies of otherwise intractable proteins.

biochemistry↗

bioRxiv2019

IL-1-conferred gene expression pattern in ERα+ BCa and AR+ PCa cells is intrinsic to ERα- BCa and AR- PCa cells and promotes cell survival

BackgroundBreast (BCa) and prostate (PCa) cancers are hormone receptor (HR)-driven cancers. Thus, BCa and PCa patients are given therapies that reduce hormone levels or directly blocks HR activity; but most patients eventually develop treatment resistance. We have previously reported that interleukin-1 (IL-1) inflammatory cytokine downregulates ER and AR mRNA in HR-positive (HR+) BCa and PCa cell lines, yet the cells can remain viable. Additionally, we identified pro-survival proteins and processes upregulated by IL-1 in HR+ BCa and PCa cells, that are basally high in HR- BCa and PCa cells. Therefore, we hypothesize that IL-1 confers a conserved gene expression pattern in HR+ BCa and PCa cells that mimics conserved basal gene expression patterns in HR- BCa and PCa cells to promote HR-independent survival and tumorigenicity.\n\nMethodsWe performed RNA sequencing (RNA-seq) for HR+ BCa and PCa cell lines exposed to IL-1 and for untreated HR- BCa and PCa cell lines. We confirmed expression patterns of select genes by RT-qPCR and used siRNA and/or drug inhibition to silence select genes in HR- BCa cell lines. Finally, we performed Ingenuity Pathway Analysis (IPA) to identify signaling pathways encoded by our RNA-seq data set.\n\nResultsWe identified 350 genes in common between BCa and PCa cells that are induced or repressed by IL-1 in HR+ cells that are, respectively, basally high or low in HR- cells. Among these genes, we identified Sequestome-1 (SQSTM1/p62) and SRY (Sex-Determining Region Y)-Box 9 (SOX9) to be essential for survival of HR- BCa and PCa cell lines. Analysis of publicly available data indicates that p62 and SOX9 expression are elevated in HR-independent BCa and PCa sublines generated in vitro, suggesting that p62 and SOX9 have a role in acquired treatment resistance. We also assessed HR- cell line viability in response to the p62-targeting drug, verteporfin, and found that verteporfin is cytotoxic for HR- cell lines.\n\nConclusionsOur 350 gene set can be used to identify novel therapeutic targets and/or biomarkers conserved among acquired (e.g. due to inflammation) or intrinsic HR-independent BCa and PCa.

cancer biology↗

bioRxiv2019

RELA is Sufficient to Mediate Interleukin-1 (IL-1) Repression of Androgen Receptor Expression and Activity in LNCaP Disease Progression Model

BackgroundThe Androgen Receptor (AR) nuclear transcription factor is a therapeutic target for prostate cancer (PCa). Unfortunately, patients can develop resistance to AR-targeted therapies and progress to lethal disease, underscoring the importance of understanding the molecular mechanisms that underlie treatment resistance. Inflammation is implicated in PCa initiation and progression and we have previously reported that the inflammatory cytokine, interleukin-1 (IL-1), represses AR mRNA levels and activity in AR-positive (AR+) PCa cell lines concomitant with the upregulation of pro-survival biomolecules. Thus, we contend that IL-1 can select for AR-independent, treatment-resistant PCa cells.\n\nMethodsTo begin to explore how IL-1 signaling leads to the repression of AR mRNA levels, we performed comprehensive pathway analysis on our RNA sequencing data from IL-1-treated LNCaP PCa cells. Our pathway analysis predicted Nuclear Factor Kappa B (NF-{kappa}B) p65 subunit (RELA), a canonical IL-1 signal transducer, to be significantly active and potentially regulate many genes, including AR. We used siRNA to silence the NF-{kappa}B family of transcription factor subunits, RELA, RELB, c-REL, NFKB1, or NFKB2, in IL-1-treated LNCaP, C4-2, and C4-2B PCa cell lines. C4-2 and C4-2B cell lines are castration-resistant LNCaP sublines and represent progression towards metastatic PCa disease; and we have previously shown that IL-1 represses AR mRNA levels in C4-2 and C4-2B cells.\n\nResultssiRNA revealed that RELA alone is sufficient to mediate IL-1 repression of AR mRNA and AR activity. Intriguingly, while LNCaP cells are more sensitive to IL-1-mediated repression of AR than C4-2 and C4-2B cells, RELA siRNA led to a more striking de-repression of AR mRNA levels and AR activity in C4-2 and C4-2B cells than in LNCaP cells.\n\nConclusionsThese data indicate that there are RELA-independent mechanisms that regulate IL-1-mediated AR repression in LNCaP cells and suggest that the switch to RELA-dependent IL-1 repression of AR in C4-2 and C4-2B cells reflects changes in epigenetic and transcriptional programs that evolve during PCa disease progression.

cancer biology↗

bioRxiv2018

A host-pathogen interaction screen identifies ada2 as a mediator of Candida glabrata defences against reactive oxygen species

Candida glabrata (C. glabrata) forms part of the normal human gut microbiota but can cause life-threatening invasive infections in immune-compromised individuals. C. glabrata displays high resistance to common azole antifungals, which necessitates new treatments. In this investigation, we identified five C. glabrata deletion mutants ({Delta}ada2, {Delta}bas1, {Delta}hir3,{Delta} ino2 and{Delta} met31) from a library of 196 transcription factor mutants that were unable to grow and activate an immune response in Drosophila larvae. This highlighted the importance of these transcription factors in C. glabrata infectivity. Further ex vivo investigation into these mutants revealed the requirement of C. glabrata ADA2 for oxidative stress tolerance. We confirmed this observation in vivo whereby growth of the C. glabrata {Delta}ada2 strain was permitted only in flies with suppressed production of reactive oxygen species (ROS). Conversely, overexpression of ADA2 promoted C. glabrata replication in infected wild type larvae resulting in larval killing. We propose that ADA2 orchestrates the response of C. glabrata against ROS-mediated immune defences during infection. With the need to find alternative antifungal treatment for C. glabrata infections, genes required for survival in the host environment, such as ADA2, provide promising potential targets.

microbiology↗