The Eyes Absent family members EYA4 and EYA1 promote PLK1 activation and successful mitosis through tyrosine dephosphorylation.

The Eyes Absent proteins (EYA1-4) are a biochemically unique group of tyrosine phosphatases known to be tumour-promoting across a range of cancer types. To date, the targets of EYA phosphatase activity remain largely uncharacterised. Here, we identify Polo-like kinase 1 (PLK1) as an interactor and phosphatase substrate of EYA4 and EYA1, with pY445 on PLK1 being the primary target site. Dephosphorylation of pY445 in the G2 phase of the cell cycle is required for centrosome maturation, PLK1 localization to centrosomes, and polo-box domain (PBD) dependent interactions between PLK1 and PLK1-activation complexes. Molecular dynamics simulations support the rationale that pY445 confers a structural impairment to PBD-substrate interactions that is relieved by EYA-mediated dephosphorylation. Depletion of EYA4 or EYA1, or chemical inhibition of EYA phosphatase activity, dramatically reduces PLK1 activation, causing mitotic defects and cell death. Overall, we have characterized a phosphotyrosine signalling network governing PLK1 and mitosis.

Dissecting the geometric and hydrophobic constraints of stapled peptides.

Stapled peptides are a promising class of molecules with potential as highly specific probes of protein-protein interactions and as therapeutics. Hydrocarbon stapling affects the peptide properties through the interplay of two factors: enhancing the overall hydrophobicity and constraining the conformational flexibility. By constructing a series of virtual peptides, we study the role of each factor in modulating the structural properties of a hydrocarbon-stapled peptide PM2, which has been shown to enter cells, engage its target Mouse Double Minute 2 (MDM2), and activate p53. Hamiltonian replica exchange molecular dynamics (HREMD) simulations suggest that hydrocarbon stapling favors helical populations of PM2 through a combination of the geometric constraints and the enhanced hydrophobicity of the peptide. To further understand the conformational landscape of the stapled peptides along the binding pathway, we performed HREMD simulations by restraining the peptide at different distances from MDM2. When the peptide approaches MDM2, the binding pocket undergoes dehydration which appears to be greater in the presence of the stapled peptide compared with the linear peptide. In the binding pocket, the helicity of the stapled peptide is increased due to the favorable interactions between the peptide residues as well as the staple and the microenvironment of the binding pocket, contributing to enhanced affinity. The dissection of the multifaceted mechanism of hydrocarbon stapling into individual factors not only deepens fundamental understanding of peptide stapling, but also provides guidelines for the design of new stapled peptides.

Development of stapled NONO-associated peptides reveals unexpected cell permeability and nuclear localisation.

The non-POU domain-containing octamer-binding protein (NONO) is a nucleic acid-binding protein with diverse functions that has been identified as a potential cancer target in cell biology studies. Little is known about structural motifs that mediate binding to NONO apart from its ability to form homodimers, as well as heterodimers and oligomers with related homologues. We report a stapling approach to macrocyclise helical peptides derived from the insulin-like growth factor binding protein (IGFBP-3) that NONO interacts with, and also from the dimerisation domain of NONO itself. Using a range of chemistries including Pd-catalysed cross-coupling, cysteine arylation and cysteine alkylation, we successfully improved the helicity and observed modest peptide binding to the NONO dimer, although binding could not be saturated at micromolar concentrations. Unexpectedly, we observed cell permeability and preferential nuclear localisation of various dye-labelled peptides in live confocal microscopy, indicating the potential for developing peptide-based tools to study NONO in a cellular context.

Transcriptional repression by a secondary DNA binding surface of DNA topoisomerase I safeguards against hypertranscription.

Regulation of global transcription output is important for normal development and disease, but little is known about the mechanisms involved. DNA topoisomerase I (TOP1) is an enzyme well-known for its role in relieving DNA supercoils for enabling transcription. Here, we report a non-enzymatic function of TOP1 that downregulates RNA synthesis. This function is dependent on specific DNA-interacting residues located on a conserved protein surface. A loss-of-function knock-in mutation on this surface, R548Q, is sufficient to cause hypertranscription and alter differentiation outcomes in mouse embryonic stem cells (mESCs). Hypertranscription in mESCs is accompanied by reduced TOP1 chromatin binding and change in genomic supercoiling. Notably, the mutation does not impact TOP1 enzymatic activity; rather, it diminishes TOP1-DNA binding and formation of compact protein-DNA structures. Thus, TOP1 exhibits opposing influences on transcription through distinct activities which are likely to be coordinated. This highlights TOP1 as a safeguard of appropriate total transcription levels in cells.

Shape complementarity processes for ultrashort-burst sensitive M13-PEG-WS2-powered MCF-7 cancer cell sensors.

Biomarkers have the potential to be utilized in disease diagnosis, prediction and monitoring. The cancer cell type is a leading candidate for next-generation biomarkers. Although traditional digital biomolecular sensor (DBS) technology has shown to be effective in assessing cell-based interactions, low cell-population detection of cancer cell types is extremely challenging. Here, we controlled the electrical signature of a two-dimensional (2D) nanomaterial, tungsten disulfide (WS2), by utilizing a combination of the Phage-integrated Polymer and the Nanosheet (PPN), viz., the integration of the M13-conjugated polyethylene glycol (PEG) and the WS2, through shape-complementarity phenomena, and developed a sensor system, i.e., the Phage-based DBS (P-DBS), for the specific, rapid, sensitive detection of clinically-relevant MCF-7 cells. The P-DBS attains a detection limit of 12 cells per µL, as well as a contrast of 1.25 between the MCF-10A sample signal and the MCF-7 sample signal. A reading length of 200 µs was further achieved, along with a relative cell viability of ∼100% for both MCF-7 and MCF-10A cells and with the PNN. Atomistic simulations reveal the structural origin of the shape complementarity-facilitated decrease in the output impedance of the P-DBS. The combination of previously unreported exotic sensing materials and digital sensor design represents an approach to unlocking the ultra-sensitive detection of cancer cell types and provides a promising avenue for early cancer diagnosis, staging and monitoring.

Success probability of high-affinity DNA aptamer generation by genetic alphabet expansion.

Nucleic acid aptamers as antibody alternatives bind specifically to target molecules. These aptamers are generated by isolating candidates from libraries with random sequence fragments, through an evolutionary engineering system. We recently reported a high-affinity DNA aptamer generation method that introduces unnatural bases (UBs) as a fifth letter into the library, by genetic alphabet expansion. By incorporating hydrophobic UBs, the affinities of DNA aptamers to target proteins are increased over 100-fold, as compared with those of conventional aptamers with only the natural four letters. However, there is still plenty of room for improvement of the methods for routinely generating high-affinity UB-containing DNA (UB-DNA) aptamers. The success probabilities of the high-affinity aptamer generation depend on the existence of the aptamer candidate sequences in the initial library. We estimated the success probabilities by analysing several UB-DNA aptamers that we generated, as examples. In addition, we investigated the possible improvement of conventional aptamer affinities by introducing one UB at specific positions. Our data revealed that UB-DNA aptamers adopt specific tertiary structures, in which many bases including UBs interact with target proteins for high affinity, suggesting the importance of the UB-DNA library design. This article is part of the theme issue 'Reactivity and mechanism in chemical and synthetic biology'.

Ultrasensitive Detection of MCF-7 Cells with a Carbon Nanotube-Based Optoelectronic-Pulse Sensor Framework.

Biosensors are of vital significance for healthcare by supporting the management of infectious diseases for preventing pandemics and the diagnosis of life-threatening conditions such as cancer. However, the advancement of the field can be limited by low sensing accuracy. Here, we altered the bioelectrical signatures of the cells using carbon nanotubes (CNTs) via structural loosening effects. Using an alternating current (AC) pulse under light irradiation, we developed a photo-assisted AC pulse sensor based on CNTs to differentiate between healthy breast epithelial cells (MCF-10A) and luminal breast cancer cells (MCF-7) within a heterogeneous cell population. We observed a previously undemonstrated increase in current contrast for MCF-7 cells with CNTs compared to MCF-10A cells with CNTs under light exposure. Moreover, we obtained a detection limit of ∼1.5 × 103 cells below a baseline of ∼1 × 104 cells for existing electrical-based sensors for an adherent, heterogeneous cell population. All-atom molecular dynamics (MD) simulations reveal that interactions between the embedded CNT and cancer cell membranes result in a less rigid lipid bilayer structure, which can facilitate CNT translocation for enhancing current. This as-yet unconsidered cancer cell-specific method based on the unique optoelectrical properties of CNTs represents a strategy for unlocking the detection of a small population of cancer cells and provides a promising route for the early diagnosis, monitoring, and staging of cancer.

Ultralong recovery time in nanosecond electroporation systems enabled by orientational-disordering processes.

The growing importance of applications based on molecular medicine and genetic engineering is driving the need to develop high-performance electroporation technologies. The electroporation phenomenon involves disruption of the cell for increasing membrane permeability. Although there is a multitude of research focused on exploring new electroporation techniques, the engineering of programming schemes suitable for these electroporation methods remains a challenge. Nanosecond stimulations could be promising candidates for these techniques owing to their ability to generate a wide range of biological responses. Here we control the membrane permeabilization of cancer cells using different numbers of electric-field pulses through orientational disordering effects. We then report our exploration of a few-volt nanosecond alternating-current (AC) stimulation method with an increased number of pulses for developing electroporation systems. A recovery time of ∼720 min was achieved, which is above the average of ∼76 min for existing electroporation methods using medium cell populations, as well as a previously unreported increased conductance with an increase in the number of pulses using weak bias amplitudes. All-atom molecular dynamics (MD) simulations reveal the orientation-disordering-facilitated increase in the degree of permeabilization. These findings highlight the potential of few-volt nanosecond AC-stimulation with an increased number of pulse strategies for the development of next-generation low-power electroporation systems.

Accelerated Ligand-Mapping Molecular Dynamics Simulations for the Detection of Recalcitrant Cryptic Pockets and Occluded Binding Sites.

The identification and characterization of binding sites is a critical component of structure-based drug design (SBDD). Probe-based/cosolvent molecular dynamics (MD) methods that allow for protein flexibility have been developed to predict ligand binding sites. However, cryptic pockets that appear only upon ligand binding and occluded binding sites with no access to the solvent pose significant challenges to these methods. Here, we report the development of accelerated ligand-mapping MD (aLMMD), which combines accelerated MD with LMMD, for the detection of these challenging binding sites. The method was validated on five proteins with what we term "recalcitrant" cryptic pockets, which are deeply buried pockets that require extensive movement of the protein backbone to expose, and three proteins with occluded binding sites. In all the cases, aLMMD was able to detect and sample the binding sites. Our results suggest that aLMMD could be used as a general approach for the detection of such elusive binding sites in protein targets, thus providing valuable information for SBDD.

An Efficient, Short Stimulus PANC-1 Cancer Cell Ablation and Electrothermal Therapy Driven by Hydrophobic Interactions.

Promising results in clinical studies have been demonstrated by the utilization of electrothermal agents (ETAs) in cancer therapy. However, a difficulty arises from the balance between facilitating the degradation of ETAs, and at the same time, increasing the electrothermal performance/stability required for highly efficient treatment. In this study, we controlled the thermal signature of the MoS2 by harnessing MoS2 nanostructures with M13 phage (MNM) via the structural assembling (hydrophobic interaction) phenomena and developed a combined PANC-1 cancer cell-MNM alternating current (AC)-stimulus framework for cancer cell ablation and electrothermal therapy. A percentage decrease in the cell viability of ~23% was achieved, as well as a degradation time of 2 weeks; a stimulus length of 100 µs was also achieved. Molecular dynamics (MD) simulations revealed the assembling kinetics in integrated M13 phage-cancer cell protein systems and the structural origin of the hydrophobic interaction-enabled increase in thermal conduction. This study not only introduced an 'ideal' agent that avoided the limitations of ETAs but also provided a proof-of-concept application of MoS2-based materials in efficacious cancer therapy.

Progressive endoplasmic reticulum stress over time due to human insulin gene mutation contributes to pancreatic beta cell dysfunction.

AIMS/HYPOTHESIS: We studied the effects of heterozygous human INS gene mutations on insulin secretion, endoplasmic reticulum (ER) stress and other mechanisms in both MIN6 and human induced pluripotent stem cells (hiPSC)-derived beta-like cells, as well as the effects of prolonged overexpression of mutant human INS in MIN6 cells. METHODS: We modelled the structure of mutant C109Y and G32V proinsulin computationally to examine the in silico effects. We then overexpressed either wild-type (WT), mutant (C109Y or G32V), or both WT and mutant human preproinsulin in MIN6 cells, both transiently and stably over several weeks. We measured the levels of human and rodent insulin secreted, and examined the transcript and protein levels of several ER stress and apoptotic markers. We also reprogrammed human donor fibroblasts heterozygous for the C109Y mutation into hiPSCs and differentiated these into pancreatic beta-like cells, which were subjected to single-cell RNA-sequencing and transcript and protein analyses for ER stress and apoptotic markers. RESULTS: The computational modelling studies, and short-term and long-term expression studies in beta cells, revealed the presence of ER stress, organelle changes and insulin processing defects, resulting in a decreased amount of insulin secreted but not the ability to secrete insulin. By 9 weeks of expression of mutant human INS, dominant-negative effects of mutant INS were evident and beta cell insulin secretory capacity declined. INS+/C109Y patient-derived beta-like cells and single-cell RNA-sequencing analyses then revealed compensatory upregulation in genes involved in insulin secretion, processing and inflammatory response. CONCLUSIONS/INTERPRETATION: The results provide deeper insights into the mechanisms of beta cell failure during INS mutation-mediated diabetes disease progression. Decreasing spliced X-box binding protein 1 (sXBP1) or inflammatory response could be avenues to restore the function of the remaining WT INS allele.

Cyclisation strategies for stabilising peptides with irregular conformations.

Cyclisation is a common synthetic strategy for enhancing the therapeutic potential of peptide-based molecules. While there are extensive studies on peptide cyclisation for reinforcing regular secondary structures such as α-helices and ß-sheets, there are remarkably few reports of cyclising peptides which adopt irregular conformations in their bioactive target-bound state. In this review, we highlight examples where cyclisation techniques have been successful in stabilising irregular conformations, then discuss how the design of cyclic constraints for irregularly structured peptides can be informed by existing ß-strand stabilisation approaches, new computational design techniques, and structural principles extracted from cyclic peptide library screening hits. Through this analysis, we demonstrate how existing peptide cyclisation techniques can be adapted to address the synthetic design challenge of stabilising irregularly structured binding motifs.

The nanotube express: Delivering a stapled peptide to the cell surface.

HYPOTHESIS: Carbon nanotubes (CNTs) represent a novel platform for cellular delivery of therapeutic peptides. Chemically-functionalized CNTs may enhance peptide uptake by improving their membrane targeting properties. EXPERIMENTS: Using coarse-grained (CG) molecular dynamics (MD) simulations, we investigate membrane interactions of a peptide conjugated to pristine and chemically-modified CNTs. As proof of principle, we focus on their interactions with PM2, an amphipathic stapled peptide that inhibits the E3 ubiquitin ligase HDM2 from negatively regulating the p53 tumor suppressor. CNT interaction with both simple planar lipid bilayers as well as spherical lipid vesicles was studied, the latter as a surrogate for curved cellular membranes. FINDINGS: Membrane permeation was rapid and spontaneous for both pristine and oxidized CNTs when unconjugated. This was slowed upon addition of a noncovalently attached peptide surface "sheath", which may be an effective way to slow CNT entry and avert membrane rupture. The CNT conjugates were observed to "desheath" their peptide layer at the bilayer interface upon insertion, leaving their cargo behind in the outer leaflet. This suggests that a synergy may exist to optimize CNT safety whilst enhancing the delivery efficiency of "hitchhiking" therapeutic molecules.

Decreased GLUT2 and glucose uptake contribute to insulin secretion defects in MODY3/HNF1A hiPSC-derived mutant ß cells.

Heterozygous HNF1A gene mutations can cause maturity onset diabetes of the young 3 (MODY3), characterized by insulin secretion defects. However, specific mechanisms of MODY3 in humans remain unclear due to lack of access to diseased human pancreatic cells. Here, we utilize MODY3 patient-derived human induced pluripotent stem cells (hiPSCs) to study the effect(s) of a causal HNF1A+/H126D mutation on pancreatic function. Molecular dynamics simulations predict that the H126D mutation could compromise DNA binding and gene target transcription. Genome-wide RNA-Seq and ChIP-Seq analyses on MODY3 hiPSC-derived endocrine progenitors reveal numerous HNF1A gene targets affected by the mutation. We find decreased glucose transporter GLUT2 expression, which is associated with reduced glucose uptake and ATP production in the MODY3 hiPSC-derived ß-like cells. Overall, our findings reveal the importance of HNF1A in regulating GLUT2 and several genes involved in insulin secretion that can account for the insulin secretory defect clinically observed in MODY3 patients.

Ultrasensitive two-dimensional material-based MCF-7 cancer cell sensor driven by perturbation processes.

Changes in lipid composition and structure during cell development can be markers for cell apoptosis or various diseases such as cancer. Although traditional fluorescence techniques utilising molecular probes have been studied, these methods are limited in studying these micro-changes as they require complex probe preparation and cannot be reused, making cell monitoring and detection challenging. Here, we developed a direct current (DC) resistance sensor based on two-dimensional (2D) molybdenum disulfide (MoS2) nanosheets to enable cancer cell-specific detection dependent on micro-changes in the cancer cell membrane. Atomistic molecular dynamics (MD) simulations were used to study the interaction between 2D MoS2 and cancer lipid bilayer systems, and revealed that previously unconsidered perturbations in the lipid bilayer can cause an increase in resistance. Under an applied DC sweep, we observed an increase in resistance when cancer cells were incubated with the nanosheets. Furthermore, a correlation was observed between the resistance and breast cancer epithelial cell (MCF-7) population, illustrating a cell population-dependent sensitivity of our method. Our method has a detection limit of ∼3 × 103 cells, below a baseline of ∼1 × 104 cells for the current state-of-the-art electrical-based biosensors using an adherent monolayer with homogenous cells. This combination of a unique 2D material and electrical resistance framework represents a promising approach for the early detection of cancerous cells and to reduce the risk of post-surgery cancer recurrence.

Macrocyclization of an all-d linear α-helical peptide imparts cellular permeability.

Peptide-based molecules hold great potential as targeted inhibitors of intracellular protein-protein interactions (PPIs). Indeed, the vast diversity of chemical space conferred through their primary, secondary and tertiary structures allows these molecules to be applied to targets that are typically deemed intractable via small molecules. However, the development of peptide therapeutics has been hindered by their limited conformational stability, proteolytic sensitivity and cell permeability. Several contemporary peptide design strategies are aimed at addressing these issues. Strategic macrocyclization through optimally placed chemical braces such as olefinic hydrocarbon crosslinks, commonly referred to as staples, may improve peptide properties by (i) restricting conformational freedom to improve target affinities, (ii) improving proteolytic resistance, and (iii) enhancing cell permeability. As a second strategy, molecules constructed entirely from d-amino acids are hyper-resistant to proteolytic cleavage, but generally lack conformational stability and membrane permeability. Since neither approach is a complete solution, we have combined these strategies to identify the first examples of all-d α-helical stapled and stitched peptides. As a template, we used a recently reported all d-linear peptide that is a potent inhibitor of the p53-Mdm2 interaction, but is devoid of cellular activity. To design both stapled and stitched all-d-peptide analogues, we used computational modelling to predict optimal staple placement. The resultant novel macrocyclic all d-peptide was determined to exhibit increased α-helicity, improved target binding, complete proteolytic stability and, most notably, cellular activity.

Straightforward Incorporation of Multiple Ligand Types into Molecular Dynamics Simulations for Efficient Binding Site Detection and Characterization.

Binding site identification and characterization is an important initial step in structure-based drug design. To account for the effects of protein flexibility and solvation, several cosolvent molecular dynamics (MD) simulation methods that incorporate small organic molecules into the protein's solvent box to probe for binding sites have been developed. However, most of these methods are highly inefficient, as they allow for the use of only one probe type at a time, which means that multiple sets of simulations have to be performed to map different types of binding sites. The high probe concentrations used in some of these methods also necessitate the use of artificial repulsive forces to prevent the probes from aggregating. Here, we present multiple-ligand-mapping MD (mLMMD), a method that incorporates multiple types of probes for simultaneous and efficient mapping of different types of binding sites without the need for introduction of artificial forces that may cause unintended mapping artifacts. We validate the method on a diverse set of 10 proteins and show that the mLMMD probes are able to reliably identify hydrophobic, hydrogen-bonding, charged, and cryptic binding sites in all of the test cases. Our results also highlight the potential utility of mLMMD for virtual screening and rational drug design.

Synthesis of Chiral Alkenyl Cyclopropane Amino Acids for Incorporation into Stapled Peptides.

α,α'-Disubstituted amino acids serve as important non-proteinogenic amino acids in the construction of stabilized helical peptides. To expand the repertoire of α,α'-disubstituted amino acids, chiral alkenyl-containing cyclopropane amino acids were synthesized via a two-step olefination and cyclopropanation procedure. Herein, we report the first example of the use of alkenyl cyclopropane building blocks to constrain MDM2-targeting helical peptides. The increased potency and efficacy associated with C-terminal cyclopropane substitution is postulated to be driven by a combined effect of net hydrophobicity and enhanced protein association rates.

Stereoisomerism of stapled peptide inhibitors of the p53-Mdm2 interaction: an assessment of synthetic strategies and activity profiles.

All-hydrocarbon, i, i+7 stapled peptide inhibitors of the p53-Mdm2 interaction have emerged as promising new leads for cancer therapy. Typical chemical synthesis via olefin metathesis results in the formation of both E- and Z-isomers, an observation that is rarely disclosed but may be of importance in targeting PPI. In this study, we evaluated the effect of staple geometry on the biological activity of five p53-reactivating peptides. We also present strategies for the modulation of the E/Z ratio and attainment of the hydrogenated adduct through repurposing of the metathesis catalyst.

Targeted covalent inhibitors of MDM2 using electrophile-bearing stapled peptides.

Herein, we describe the development of a novel staple with an electrophilic warhead to enable the generation of stapled peptide covalent inhibitors of the p53-MDM2 protein-protein interaction (PPI). The peptide developed showed complete and selective covalent binding resulting in potent inhibition of p53-MDM2 PPI.