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Targeted degradation approaches such as proteolysis targeting chimeras (PROTACs) offer new ways to address disease through tackling challenging targets and with greater potency, efficacy, and specificity over traditional approaches. However, identification of high-affinity ligands to serve as PROTAC starting points remains challenging. As a complementary approach, we describe a class of molecules termed biological PROTACs (bioPROTACs)—engineered intracellular proteins consisting of a target-binding domain directly fused to an E3 ubiquitin ligase. 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. We then identified a highly effective bioPROTAC against an oncology target, proliferating cell nuclear antigen (PCNA) to elicit rapid and robust PCNA degradation and associated effects on DNA synthesis and cell cycle progression. Overall, bioPROTACs are powerful tools for interrogating degradation approaches, target biology, and potentially for making therapeutic impacts.

Although stapled α-helical peptides can address challenging targets, their advancement is impeded by poor understandings for making them cell permeable while avoiding off-target toxicities. By synthesizing >350 molecules, we present workflows for identifying stapled peptides against Mdm2(X) with in vivo activity and no off-target effects. Key insights include a clear correlation between lipophilicity and permeability, removal of positive charge to avoid off-target toxicities, judicious anionic residue placement to enhance solubility/behavior, optimization of C-terminal length/helicity to enhance potency, and optimization of staple type/number to avoid polypharmacology. Workflow application gives peptides with >292x improved cell proliferation potencies and no off-target cell proliferation effects ( > 3800x on-target index). Application of these ‘design rules’ to a distinct Mdm2(X) peptide series improves ( > 150x) cellular potencies and removes off-target toxicities. The outlined workflow should facilitate therapeutic impacts, especially for those targets such as Mdm2(X) that have hydrophobic interfaces and are targetable with a helical motif. Stapled α-helical peptides are promising for targeting challenging targets such as transcription factors, but achieving sufficient cell permeability while avoiding off-target cleavage is difficult. Here, the authors present workflows for identifying stapled peptides against Mdm2(X) with in vivo activity and no off-target effects based on comprehensive investigations of their properties.

Stapled α-helical peptides represent an emerging superclass of macrocyclic molecules with drug-like properties, including high-affinity target binding, protease resistance, and membrane permeability. As a model system for probing the chemical space available for optimizing these properties, we focused on dual Mdm2/MdmX antagonist stapled peptides related to the p53 N-terminus. Specifically, we first generated a library of ATSP-7041 (Chang et al., 2013) analogs iteratively modified by L-Ala and D-amino acids. Single L-Ala substitutions beyond the Mdm2/(X) binding interfacial residues (i.e., Phe3, Trp7, and Cba10) had minimal effects on target binding, α-helical content, and cellular activity. Similar binding affinities and cellular activities were noted at non-interfacial positions when the template residues were substituted with their d-amino acid counterparts, despite the fact that d-amino acid residues typically ‘break’ right-handed α-helices. d-amino acid substitutions at the interfacial residues Phe3 and Cba10 resulted in the expected decreases in binding affinity and cellular activity. Surprisingly, substitution at the remaining interfacial position with its d-amino acid equivalent (i.e., Trp7 to d-Trp7) was fully tolerated, both in terms of its binding affinity and cellular activity. An X-ray structure of the d-Trp7-modified peptide was determined and revealed that the indole side chain was able to interact optimally with its Mdm2 binding site by a slight global re-orientation of the stapled peptide. To further investigate the comparative effects of d-amino acid substitutions we used linear analogs of ATSP-7041, where we replaced the stapling amino acids by Aib (i.e., R84 to Aib4 and S511 to Aib11) to retain the helix-inducing properties of α-methylation. The resultant analog sequence Ac–Leu–Thr–Phe–Aib–Glu–Tyr–Trp–Gln–Leu–Cba–Aib–Ser–Ala–Ala–NH2 exhibited high-affinity target binding (Mdm2 Kd = 43 nM) and significant α-helicity in circular dichroism studies. Relative to this linear ATSP-7041 analog, several d-amino acid substitutions at Mdm2(X) non-binding residues (e.g., d-Glu5, d-Gln8, and d-Leu9) demonstrated decreased binding and α-helicity. Importantly, circular dichroism (CD) spectroscopy showed that although helicity was indeed disrupted by d-amino acids in linear versions of our template sequence, stapled molecules tolerated these residues well. Further studies on stapled peptides incorporating N-methylated amino acids, l-Pro, or Gly substitutions showed that despite some positional dependence, these helix-breaking residues were also generally tolerated in terms of secondary structure, binding affinity, and cellular activity. Overall, macrocyclization by hydrocarbon stapling appears to overcome the destabilization of α-helicity by helix breaking residues and, in the specific case of d-Trp7-modification, a highly potent ATSP-7041 analog (Mdm2 Kd = 30 nM; cellular EC50 = 600 nM) was identified. Our findings provide incentive for future studies to expand the chemical diversity of macrocyclic α-helical peptides (e.g., d-amino acid modifications) to explore their biophysical properties and cellular permeability. Indeed, using the library of 50 peptides generated in this study, a good correlation between cellular permeability and lipophilicity was observed.

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 (IFNγ) response. Genetic deletion of STUB1 increased IFNGR1 abundance on the cell surface and thus enhanced the downstream IFNγ 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 IFNγ sensing, prompting further investigations to assess if broader inactivation of human STUB1 in both tumors and immune cells could overcome ICB resistance.

Recent COVID-19 vaccines unleashed the potential of mRNA-based therapeutics. A common bottleneck across mRNA-based therapeutic approaches is the rapid design of mRNA sequences that are translationally efficient, long-lived and non-immunogenic. Currently, an accessible software tool to aid in the design of such high-quality mRNA is lacking. Here, we present mRNAid, an open-source platform for therapeutic mRNA optimization, design and visualization that offers a variety of optimization strategies for sequence and structural features, allowing one to customize desired properties into their mRNA sequence. We experimentally demonstrate that transcripts optimized by mRNAid have characteristics comparable with commercially available sequences. To encompass additional aspects of mRNA design, we experimentally show that incorporation of certain uridine analogs and untranslated regions can further enhance stability, boost protein output and mitigate undesired immunogenicity effects. Finally, this study provides a roadmap for rational design of therapeutic mRNA transcripts.

NRC publication: Yes

Cyclic peptides are poised to target historically difficult to drug intracellular protein–protein interactions, however, their general cell impermeability poses a challenge for characterizing function. Recent advances in microfluidics have enabled permeabilization of the cytoplasmic membrane by physical cell deformation (i.e., mechanoporation), resulting in intracellular delivery of impermeable macromolecules in vector‐ and electrophoretic‐free approaches. However, the number of payloads (e.g., peptides) and/or concentrations delivered via microfluidic mechanoporation is limited by having to pre‐mix cells and payloads, a manually intensive process. In this work, we show that cells are momentarily permeable ( t 1/2  = 1.1–2.8 min) after microfluidic vortex shedding (μVS) and that lower molecular weight macromolecules can be cytosolically delivered upon immediate exposure after cells are processed/permeabilized. To increase the ability to screen peptides, we built a system, dispensing‐microfluidic vortex shedding (DμVS), that integrates a μVS chip with inline microplate‐based dispensing. To do so, we synced an electronic pressure regulator, flow sensor, on/off dispense valve, and an x‐y motion platform in a software‐driven feedback loop. Using this system, we were able to deliver low microliter‐scale volumes of transiently mechanoporated cells to hundreds of wells on microtiter plates in just several minutes (e.g., 96‐well plate filled in <2.5 min). We validated the delivery of an impermeable peptide directed at MDM2, a negative regulator of the tumor suppressor p53, using a click chemistry‐ and NanoBRET‐based cell permeability assay in 96‐well format, with robust delivery across the full plate. Furthermore, we demonstrated that DμVS could be used to identify functional, low micromolar, cellular activity of otherwise cell‐inactive MDM2‐binding peptides using a p53 reporter cell assay in 96‐ and 384‐well format. Overall, DμVS can be combined with downstream cell assays to investigate intracellular target engagement in a high‐throughput manner, both for improving structure–activity relationship efforts and for early proof‐of‐biology of non‐optimized peptide (or potentially other macromolecular) tools.

Proteins that limit the activity of the tumour suppressor protein p53 are increasingly being targeted for inhibition in a variety of cancers. In addition to the development of small molecules, there has been interest in developing constrained (stapled) peptide inhibitors. A stapled peptide ALRN_6924 that activates p53 by preventing its interaction with its negative regulator Mdm2 has entered clinical trials. This stapled peptide mimics the interaction of p53 with Mdm2. The chances that this peptide could bind to other proteins that may also interact with the Mdm2-binding region of p53 are high; one such protein is the CREB binding protein (CBP)/p300. It has been established that phosphorylated p53 is released from Mdm2 and binds to p300, orchestrating the transcriptional program. We investigate whether molecules such as ALRN_6924 would bind to p300 and, to do so, we used molecular simulations to explore the binding of ATSP_7041, which is an analogue of ALRN_6924. Our study shows that ATSP_7041 preferentially binds to Mdm2 over p300; however, upon phosphorylation, it appears to have a higher affinity for p300. This could result in attenuation of the amount of free p300 available for interacting with p53, and hence reduce its transcriptional efficacy. Our study highlights the importance of assessing off-target effects of peptide inhibitors, particularly guided by the understanding of the networks of protein-protein interactions (PPIs) that are being targeted.

PurposeTo develop a novel, target agnostic liposome click membrane permeability assay (LCMPA) using liposome encapsulating copper free click reagent dibenzo cyclooctyne biotin (DBCO-Biotin) to conjugate azido modified peptides that may effectively translocate from extravesicular space into the liposome lumen.MethodDBCO-Biotin liposomes were prepared with egg phosphatidylcholine and cholesterol by lipid film rehydration, freeze/thaw followed by extrusion. Size of DBCO-Biotin liposomes were characterized with dynamic light scattering.ResultsThe permeable peptides representing energy independent mechanism of permeability showed higher biotinylation in LCMPA. Individual peptide permeability results from LCMPA correlated well with shifts in potency in cellular versus biochemical assays (i.e., cellular/ biochemical ratio) demonstrating quantitative correlation to intracellular barrier in intact cells.ConclusionThe study provides a novel membrane permeability assay that has potential to evaluate energy independent transport of diverse peptides.