Biology subjects

Partridge, A. W.

Publications and source records attributed to Partridge, A. W..

9 recordsLinked to original sources

bioRxiv2026

Mirror-image mRNA display uncovers isoform-selective D-peptide macrocycles targeting a cryptic KRAS pocket

Activating KRAS mutations drive millions of cancers diagnosed worldwide,1 yet for decades this oncoprotein was deemed "undruggable", reflecting the challenge of discovering molecules capable of perturbing its complex biological functions, and of translating these discoveries into effective cancer therapeutics.2 Recent advances propelled by innovative screening have identified diverse modalities that bind at or near the switch-II pocket (SII-P) of RAS proteins, including molecular glues,3 macrocyclic peptides,4 fragment-derived small molecules,5 and approved therapies that covalently target KRASG12C.6,7 Unfortunately, resistance to approved therapies has emerged,8,9 highlighting the need for molecules that engage new or underexploited binding sites on RAS oncoproteins with mechanisms complementary to established SII-P inhibitors.10,11 Here we show that mirror-image mRNA display12 enabled the discovery of all-D macrocyclic peptide ligands targeting a cryptic RAS back pocket (CRB-P).13 These ligands engage KRAS(OFF) and KRAS(ON) with equal affinity, exploit a single-residue difference among isoforms to bind KRAS selectively, and successfully inhibit oncogenic signaling in KRAS-mutant cells through a mechanism distinct from SII-P binders. Mirror-image screening directly afforded nanomolar peptide ligands stable toward cellular proteolysis and delivered probes targeting distinct epitopes not accessible by homochiral peptide-display methods. Together, these findings establish the CRB-P as a specifically druggable and mechanistically differentiated site on KRAS with potential for combination with emerging RAS-targeting therapies and substantiate mirror-image mRNA display as a strategy for discovering stable all-D macrocyclic peptides targeting previously inaccessible epitopes on challenging targets.

cancer biology↗

bioRxiv2023

Design-Rules for Stapled Alpha-Helical Peptides with On-Target In Vivo Activity: Application toMdm2/X dual antagonists

Stapled -helical peptides can bind to and modulate historically intractable targets while addressing the traditional liabilities associated with peptide therapeutics. However, their pipeline advancement has been impeded by the challenges of identifying peptides with sufficient cellular uptake to engage the target protein while lacking off-target toxicities. Here, we advance the field to arrive at a workflow for identifying advanced stapled peptide lead molecules with on-target in vivo activity with no off-target cell proliferation effects. Specifically, we generated a >350-member library based on ATSP-7041, a stapled peptide Mdm2(X) antagonist with validated on-target cellular effects but with significant off-target activity. Key insights from library analysis include 1) a clear correlation between lipophilicity and permeability, 2) removal of positive charge to avoid off-target toxicities, 3) judicious placement of anionic residues to enhance peptide solubility/behavior, 4) optimization of C-terminal length and helicity to enhance cell activity, 5) optimization of staple type/number to avoid polypharmacology. Incorporation of one or more of these attributes led to molecules with improved in vitro and in vivo activities (up to a >292x improved cell proliferation EC50). A subset of peptides were devoid of off-target cell proliferation effects in cell lines lacking wild-type p53 protein (up to a >3800x on-target index). This latter improvement contrasted with clinical Mdm2 antagonistic molecules. Application of these design rules to a distinct Mdm2(X) peptide series resulted in rapid improvement in cellular activity (>150x) and removal of off-target toxicities. Overall, the detailed workflow outlined here should help researchers identify stapled -helical peptides for therapeutic impact.

biochemistry↗

bioRxiv2022

mRNAid, an Open-Source Platform for Therapeutic mRNA Design and Optimization Strategies

Recent COVID-19 vaccines unleashed the potential of mRNA-based therapeutics. mRNA optimization is indispensable for reducing immunogenicity, ensuring stability, and maximizing protein output. We present mRNAid, an experimentally validated software for mRNA optimization and visualization that generates mRNA sequences with comparable if not superior characteristics to commercially optimized sequences. To encompass all aspects of mRNA design, we also interrogated the impact of uridine content, nucleoside analogs and UTRs on expression and immunogenicity.

bioinformatics↗

bioRxiv2022

Targeted degradation of PCNA outperforms stoichiometric inhibition to result in programed cell death

Targeted protein degradation has emerged as a powerful technology - both as a biological tool and for broadening the therapeutic proteome. As tools to probe this approach on historically intractable targets, we have previously advanced biodegraders -- targeted degradation fusion constructs composed of mini-proteins/peptides linked to modified E3 ligase receptors. Herein, we gain deeper insights into the utility and potential of biodegraders, through a detailed study on Con1-SPOP, a biodegrader which rapidly degrades the potential cancer target, proliferating cell nuclear antigen (PCNA). In a variety of settings, the active biodegrader (Con1-SPOP) proved pharmacologically superior to its stoichiometric (non-degrading) inhibitor equivalent (Con1-SPOPmut). Specifically, in addition to more potent anti-proliferative effects in both 2D cell culture and 3D spheroids, PCNA degradation uniquely induced DNA damage, cell apoptosis and necrosis. Global proteomic profiling of a stable cell-line expressing Con1-SPOP under doxycycline (Dox) induction revealed that impaired mitotic division and mitochondria dysfunction is a direct consequence of PCNA degradation, effects not seen with the stoichiometric inhibitor protein. To evaluate the therapeutic potential of biodegraders, we showed that Dox-induced Con1-SPOP achieved complete tumor-growth inhibition in a xenograft model. To explore application of biodegraders as a novel therapeutic modality, modified mRNA encoding Con1-SPOP was synthesized and encapsulated into lipid nanoparticles (LNPs). The approach successfully delivered mRNA in vitro to deplete endogenous PCNA within hours of application and with nanomolar potency. Overall, our results demonstrate the utility of biodegraders as biological tools and highlight target-degradation as a more efficacious approach versus stoichiometric inhibition. Finally, once in vivo delivery and expression are optimized, biodegraders may be leveraged as an exciting therapeutic modality.

cell biology↗

bioRxiv2021

Discovery of cell active macrocyclic peptides with on-target inhibition of KRAS signaling

RAS is the most commonly mutated oncogene in human cancers and RAS-driven tumors are amongst the most difficult to treat. Historically, discovery of therapeutics targeting this protein has proven challenging due to a lack of deep hydrophobic pockets to which a small molecule might bind. The single such pocket available is normally occupied by GDP or GTP which have millimolar cellular concentrations and picomolar affinities for KRAS and hence is challenging to target. The recent FDA approval of sotorasib, a covalent modifier of the KRASG12C mutant protein, has clinically validated KRAS as an oncology target. However, traditional challenges remain for targeting the more common KRAS mutations such as G12D and G12V. As an alternative approach, we investigated the superior binding capacity of macrocyclic peptides to identify KRAS inhibitory molecules. We focused on the recently reported disulfide-cyclized arginine-rich peptide KRpep-2d, discovered through phage display and previously independently confirmed by us as a bona fide KRAS binder. To mitigate intracellular disulfide reduction and loss of binding, we identified an alternate cyclization motif by inverting the configuration of Cys5 and linking it to Cys15 through a thioacetal bridge. The corresponding peptide bound KRAS through cis isomerization of the peptide bond between D-Cys5 and Pro6 as observed through x-ray crystallography. Prototypic molecules displayed measurable cellular inhibition of RAS signaling without membrane lysis and counter-screen off-target activity. An analogue containing azido-lysine confirmed the cell penetrant nature of this molecule using our recently reported NanoClick assay. To improve cellular activity, we sought to improve proteolytic stability. Structure-activity relationship studies identified key scaffold residues critical for binding and revealed that replacement of N- and C-terminal arginine residues with D-arginines and introduction of an -methyl moiety at Ser10 resulted in a molecule with improved proteolytic stability as indicated by its persistence in whole cell homogenate. The resulting peptide MP-3995 had improved and sustained cell-based efficacy. On-target activity was validated through confirmation of target engagement, lack of signaling in irrelevant pathways, and use of non-binding control peptides. Mechanism of action studies suggested that peptide binding to both GDP- and GTP-states of KRAS may contribute to cellular activity. Although validated as bona fide and on-target inhibitors of cell based KRAS signaling, this series is unlikely to advance to the clinic in its current form due to its arginine-dependent cell entry mechanism. Indeed, we showed a strong correlation between net positive charge and histamine release in an ex vivo assay making this series challenging to study in vivo. Nonetheless, these molecules provide valuable templates for further medicinal chemistry efforts aimed at leveraging this unique inhibitory binding site on KRAS. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/459913v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@17f4764org.highwire.dtl.DTLVardef@12818daorg.highwire.dtl.DTLVardef@177bdf0org.highwire.dtl.DTLVardef@57046c_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2020

STUB1 is an intracellular checkpoint for interferon gamma sensing

Immune checkpoint blockade (ICB) leads to durable and complete tumour regression in some patients but in others gives temporary, partial or no response. Accordingly, significant efforts are underway to identify tumour-intrinsic mechanisms underlying ICB resistance. Results from a published CRISPR screen in a mouse model suggested that targeting STUB1, an E3 ligase involved in protein homeostasis, may overcome ICB resistance but the molecular basis of this effect remains unclear. Herein, we report an under-appreciated role of STUB1 to dampen the interferon gamma (IFN{gamma}) response. Genetic deletion of STUB1 increased IFNGR1 abundance on the cell surface and thus enhanced the downstream IFN{gamma} response as showed by multiple approaches including Western blotting, flow cytometry, qPCR, phospho-STAT1 assay, immunopeptidomics, proteomics, and gene expression profiling. Human prostate and breast cancer cells with STUB1 deletion were also susceptible to cytokine-induced growth inhibition. Furthermore, blockade of STUB1 protein function recapitulated the STUB1-null phenotypes. Despite these encouraging in vitro data and positive implications from clinical datasets, we did not observe in vivo benefits of inactivating Stub1 in mouse syngeneic tumour models - with or without combination with anti-PD-1 therapy. However, our findings elucidate STUB1 as a barrier to IFN{gamma} sensing, prompting further investigations to assess if broader inactivation of human STUB1 in both tumors and immune cells could overcome ICB resistance.

cancer biology↗

bioRxiv2020

bioPROTACs establish RAS as a degradable target and provide novel RAS biology insights

ABSTRACTMutations to RAS proteins (H-, N-, and K-RAS) are amongst the most common oncogenic drivers and tumors harboring these lesions are some of the most difficult to treat. Although the recently discovered covalent small molecules against the KRASG12C mutant have shown promising efficacy against lung cancers, traditional barriers remain for drugging the more prevalent KRASG12D and KRASG12V mutants. Targeted degradation has emerged as an attractive alternative approach but for KRAS, identification of the required high-affinity ligands continues to be a challenge. Another significant hurdle is the discovery of a hybrid molecule that appends an E3 ligase-recruiting moiety in a manner that satisfies the precise geometries required for productive polyubiquitin transfer while maintaining favorable drug-like properties. As a tool to gain insights into the advantages and feasibility of KRAS targeted-degradation, we applied the bioPROTAC approach. This workflow centers on the intracellular expression of a chimeric protein consisting of a high-affinity target-binding domain fused to an engineered E3 ligase adapter. We generated a series of anti-RAS bioPROTACs that span different RAS isoform/nucleotide-state specificities and leverage different E3 ligases. Overall, our results provide definitive evidence for the degradability of RAS proteins. We further elucidate the functional consequences of RAS degradation, the susceptibility and degradation kinetics of various mutant KRAS, and the prevalence of different nucleotide-states in WT and mutant KRAS. Finally, if delivery challenges can be addressed, anti-RAS bioPROTACs will be exciting candidates for clinical development.Competing Interest StatementThe authors have declared no competing interest.View Full Text

cancer biology↗

bioRxiv2019

bioPROTACs as versatile modulators of intracellular therapeutic targets: Application to proliferating cell nuclear antigen (PCNA)

Targeted degradation approaches have recently generated much excitement as a paradigm shift to address human disease in unprecedented ways. Amongst these, small molecule based approaches such as Proteolysis targeting chimeras (PROTACs) have attracted the lions share of attention due to their potential to tackle historically intractable targets and achieve greater potency, efficacy, and specificity over traditional small molecule inhibitors. Despite their promise, the identification of high-affinity ligands that can serve as starting points for PROTAC strategies remains challenging. As a complementary approach, we describe herein a class of intracellular biologics termed bioPROTACs. The substrate binding component of these fusion proteins consists of a peptide or an antibody-mimetic which allows for an unprecedented diversity of protein targets that can be addressed. The high-affinity binder is linked directly to an E3 ubiquitin ligase to harness the power of targeted degradation. Using GFP-tagged proteins as model substrates, we show that there is considerable flexibility in both the choice of substrate binders (binding positions, scaffold-class) and the E3 ligases. Indeed, 9 out of 16 binder-E3 combinations tested resulted in greater than 70% target clearance. Through a systematic approach, we then identified a highly effective bioPROTAC against an oncology target, proliferating cell nuclear antigen (PCNA), a sliding DNA clamp with critical roles in DNA replication and repair. The bioPROTAC, termed Con1-SPOP, elicited rapid and robust PCNA degradation and associated effects on DNA synthesis and cell cycle progression. Compared to RNAi-based approaches which typically take days to manifest, PCNA knockdown using Con1-SPOP was evident within 4 h. The advantage of degradation versus stoichiometric inhibition was also clearly demonstrated with bioPROTAC strategies. Combining superior pharmacological inhibition and relative ease of development, bioPROTACs are powerful tools for interrogating the degradability of a substrate, for guiding the identification of the fittest E3 ligase, for studying the functional consequences associated with target protein down-regulation, and potentially for making therapeutic impacts.

cell biology↗

bioRxiv2019

De-risking drug discovery of intracellular targeting peptides: screening strategies to eliminate false-positive hits

Discovery of false-positive target binding, due to assay interference or aggregation, presents a significant problem for drug discovery programs. These issues may often be unrealized and could lead researchers astray if not subject to independent verification of reproducibility and/or on-target mechanism of action. Although well-documented for small molecules, this issue has not been widely explored for peptide modality. As a case study, we demonstrate that two purported KRas inhibitors, stapled peptide SAH-SOS1A and macrocyclic peptide cyclorasin 9A5, exemplify false-positive molecules - both in terms of their sub-micromolar KRas binding affinities and their on-target cellular activities. We observed that the apparent binding of fluorescein-labeled SAH-SOS1A given by a fluorescence polarization assay is sensitive to detergent. False-positive readouts can arise from peptide adsorption to the surface of microplates. Hence, we used surface plasmon resonance and isothermal titration calorimetry to unambiguously show that both SAH-SOS1A and cyclorasin 9A5 are non-binders for KRas. Thermal shift assay and hydrogen-deuterium exchange mass spectrometry further demonstrate that both peptides destabilize KRas and induce unfolding of the protein. Furthermore, both peptides caused significant release of intracellular lactate dehydrogenase, suggesting that membrane rupture rather than on-target activity is accountable for their reported cytotoxicity. Finally, both peptides exhibited off-target activities by inhibiting the proliferation of U-2 OS and A549 cells, despite their independency of the KRas signaling pathway. Our findings demonstrate the critical need to employ orthogonal binding assays and cellular counter-screens to de-risk false-positive molecules. More rigorous workflows should lead to improved data and help obviate inadvertent scientific conclusions.\n\nSignificance statementFalse positive molecule hits occur frequently in high-throughput screens and can contaminate the scientific literature. This has become an increasingly serious issue in small molecule drug discovery and chemical probe development and it is not surprising that peptides may be similarly prone to assay interference. Using KRas as a target and two known macrocyclic peptide inhibitors as a case study, we clearly show that reporter-free biophysical assays and cellular counter-screens offer the solution to detect and de-risk the potential of false-positive compounds. We further discuss the advantages, limitations and overall strategic importance of such methods.

biochemistry↗