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Abstract 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.

Abstract 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 (t1/2 = 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

Approaches have been devised to increase the discovery rate of intrabodies but often these yield results that aren’t functional in cells. Here the authors engineer and optimise an autonomous and disulphide-free human VH domain for intracellular expression, and they identify several VH domain binders against eIF4E.

“Managed Aquifer Recharge: A Key to Sustainability” is the title of the fourth Special Issue presented by the journal Water (MDPI), dedicated to the 11th International Symposium on Managed Aquifer Recharge (ISMAR 11) that was held between 11 and 15 April 2022 in Long Beach, California, USA [...]

RadH is one of the flavin-dependent halogenases that has previously exhibited promising catalytic activity towards hydroxycoumarin, hydroxyisoquinoline, and phenolic derivatives. Here, we evaluated new functional homologs of RadH and expanded its specificities for the halogenation of non-tryptophan-derived, heterocyclic scaffolds. Our investigation revealed that RadH could effectively halogenate hydroxyquinoline and hydroxybenzothiophene. Assay optimization studies revealed the need to balance the various co-factor concentrations and where a GDHi co-factor recycling system most significantly improves the conversion and efficiency of the reaction. A crystal structure of RadH was also obtained with a resolution of 2.4 Å, and docking studies were conducted to pinpoint the binding and catalytic sites for substrates.

Coastal ecosystems such as those in the Great Barrier Reef (GBR) lagoon, are exposed to stressors in flood plumes including low light (caused by increased turbidity) and agricultural pesticides. Photosystem II (PSII)-inhibiting herbicides are the most frequently detected pesticides in the GBR lagoon, but it is not clear how their toxicity to phototrophic species depends on light availability. This study investigated the individual and combined effects of PSII-inhibiting herbicide, diuron, and reduced light intensity (as a proxy for increased turbidity) on the marine diatom, Phaeodactylum tricornutum. Effective quantum yield (EQY) and cell density were measured to calculate responses relative to the controls over 72-h, in tests with varying stressor intensities. Individually, diuron concentrations (0.1–3 μg l−1) were not high enough to significantly reduce growth (cell density), but led to decreased EQY; while, low light generally led to increased EQY, but only reduced growth at the lowest tested light intensity (5 μmol photons m−2 s−1) after 48-hours. P. tricornutum was less affected by diuron when combined with low light scenarios, with increased EQY (up to 163% of the controls) that was likely due to increased electron transport per photon, despite lesser available photons at this low light intensity. In contrast, growth was completely inhibited relative to the controls when algae were simultaneously exposed to the highest stressor levels (3 μg l−1 diuron and 5 μmol photons m−2 s−1). This study highlights the importance of measuring more than one biological response variable to capture the combined effects of multiple stressors. Management of water quality stressors should consider combined impacts rather than just the impacts of individual stressors alone. Reducing suspended sediment and diuron concentrations in marine waters can decrease harmful effects and bring synergistic benefits to water quality.

Abstract Animal movement studies are conducted to monitor ecosystem health, understand ecological dynamics, and address management and conservation questions. In marine environments, traditional sampling and monitoring methods to measure animal movement are invasive, labor intensive, costly, and limited in the number of individuals that can be feasibly tracked. Automated detection and tracking of small‐scale movements of many animals through cameras are possible but are largely untested in field conditions, hampering applications to ecological questions. Here, we aimed to test the ability of an automated object detection and object tracking pipeline to track small‐scale movement of many individuals in videos. We applied the pipeline to track fish movement in the field and characterize movement behavior. We automated the detection of a common fisheries species (yellowfin bream, Acanthopagrus australis) along a known movement passageway from underwater videos. We then tracked fish movement with three types of tracking algorithms (MOSSE, Seq‐NMS, and SiamMask) and evaluated their accuracy at characterizing movement. We successfully detected yellowfin bream in a multispecies assemblage (F1 score =91%). At least 120 of the 169 individual bream present in videos were correctly identified and tracked. The accuracies among the three tracking architectures varied, with MOSSE and SiamMask achieving an accuracy of 78% and Seq‐NMS 84%. By employing this integrated object detection and tracking pipeline, we demonstrated a noninvasive and reliable approach to studying fish behavior by tracking their movement under field conditions. These cost‐effective technologies provide a means for future studies to scale‐up the analysis of movement across many visual monitoring systems.

Abstract Connectivity is fundamentally important for shaping the resilience of complex human and natural networks when systems are disturbed. Ecosystem resilience is, in part, shaped by the spatial arrangement of habitats, the permeability and fluxes between them, the stabilising functions performed by organisms, their dispersal traits, and the interactions between functions and stressor types. Controlled investigations of the relationships between these phenomena under multiple stressors are sparse, possibly due to logistic and ethical difficulties associated with applying and controlling stressors at landscape scales. Here we show that grazing performance, a key ecosystem function, is linked to connectivity by manipulating the spatial configuration of habitats in microcosms impacted by multiple stressors. Greater connectivity enhanced ecosystem function and reduced variability in grazing performance in unperturbed systems. Improved functional performance was observed in better connected systems stressed by harvesting pressure and temperature rise, but this effect was notably reversed by the spread of disease. Connectivity has complex effects on ecological functions and resilience, and the nuances should be recognised more fully in ecosystem conservation.

Abstract Background Precision therapeutics continuously make advances in cancer therapy, and a field of growing interest is the combination of targeted radionuclide therapy (TRNT) with potential radiosensitizing agents. This study evaluated whether the effects of in vitro TRNT, using the 177Lu-labeled anti-CD44v6 antibody AbN44v6, were potentiated by the novel stapled MDM2/X-p53 antagonist PM2. Materials and methods Two wt p53 cell lines, HCT116 (colorectal carcinoma) and UM-SCC-74B (head and neck squamous cell carcinoma), expressing different levels of the target antigen, CD44v6, were used. Antigen-specific binding of 177Lu-AbN44v6 was initially verified in a 2D cell assay, after which the potential effects of unlabeled AbN44v6 on downstream phosphorylation of Erk1/2 were evaluated by western blotting. Further, the therapeutic effects of unlabeled AbN44v6, 177Lu-AbN44v6, PM2, or a combination (labeled/unlabeled AbN44v6 +/− PM2) were assessed in 3D multicellular tumor spheroid assays. Results Radiolabeled antibody bound specifically to CD44v6 on both cell lines. Unlabeled AbN44v6 binding did not induce downstream phosphorylation of Erk1/2 at any of the concentrations tested, and repeated treatments with the unlabeled antibody did not result in any spheroid growth inhibition. 177Lu-AbN44v6 impaired spheroid growth in a dose-dependent and antigen-dependent manner. A single modality treatment with 20 μM of PM2 significantly impaired spheroid growth in both spheroid models. Furthermore, the combination of TRNT and PM2-based therapy proved significantly more potent than either monotherapy. In HCT116 spheroids, this resulted in a two- and threefold spheroid growth rate decrease for the combination of PM2 and 100 kBq 177Lu-AbN44v6 compared to monotherapies 14-day post treatment. In UM-SCC-74B spheroids, the combination therapy resulted in a reduction in spheroid size compared to the initial spheroid size 10-day post treatment. Conclusion TRNT using 177Lu-AbN44v6 proved efficient in stalling spheroid growth in a dose-dependent and antigen-dependent manner, and PM2 treatment demonstrated a growth inhibitory effect as a monotherapy. Moreover, by combining TRNT with PM2-based therapy, therapeutic effects of TRNT were potentiated in a 3D multicellular tumor spheroid model. This proof-of-concept study exemplifies the strength and possibility of combining TRNT targeting CD44v6 with PM2-based therapy.

Global-scale conservation initiatives and policy instruments rely on ecosystem indicators to track progress towards targets and objectives. A deeper understanding of indicator interrelationships would benefit these efforts and help characterize ecosystem status. We study interrelationships among 34 indicators for mangroves, saltmarsh, and seagrass ecosystems, and develop data-driven, spatially explicit typologies of coastal wetland status at a global scale. After accounting for environmental covariates and gap-filling missing data, we obtained two levels of clustering at 5 and 18 typologies, providing outputs at different scales for different end users. We generated 2,845 cells (1° (lat) × 1° (long)) globally, of which 29.7% were characterized by high land- and marine-based impacts and a high proportion of threatened species, 13.5% by high climate-based impacts, and 9.6% were refuges with lower impacts, high fish density and a low proportion of threatened species. We identify instances where specific actions could have positive outcomes for coastal wetlands across regions facing similar issues. For example, land- and marine-based threats to coastal wetlands were associated with ecological structure and function indicators, suggesting that reducing these threats may reduce habitat degradation and threats to species persistence. However, several interdimensional relationships might be affected by temporal or spatial mismatches in data. Weak relationships mean that global biodiversity maps that categorize areas by single indicators (such as threats or trends in habitat size) may not be representative of changes in other indicators (e.g., ecosystem function). By simplifying the complex global mosaic of coastal wetland status and identifying regions with similar issues that could benefit from knowledge exchange across national boundaries, we help set the scene for globally and regionally coordinated conservation.

Vegetated coastal wetlands, including seagrass, saltmarsh and mangroves, are threatened globally, yet the need to avert these losses is poorly recognized in international policy, such as in the Convention on Biological Diversity and the United Nations (UN) Sustainable Development Goals. Identifying the impact of overlooking coastal wetlands in ecosystem assessment frameworks could help prioritize research efforts to fill these gaps. Here, we examine gaps in the recognition of coastal wetlands in globally applicable ecosystem assessments. We address both shortfalls in assessment frameworks when it comes to assessing wetlands, and gaps in data that limit widespread application of assessments. We examine five assessment frameworks that track fisheries, greenhouse gas emissions, ecosystem threats, and ecosystem services. We found that these assessments inform management decisions, but that the functions provided by coastal wetlands are incompletely represented. Most frameworks had sufficient complexity to measure wetland status, but limitations in data meant they were incompletely informed about wetland functions and services. Incomplete representation of coastal wetlands may lead to them being overlooked by research and management. Improving the coverage of coastal wetlands in ecosystem assessments requires improving global scale mapping of wetland trends, developing global-scale indicators of wetland function and synthesis to quantitatively link animal population dynamics to wetland trends. Filling these gaps will help ensure coastal wetland conservation is properly informed to manage them for the outstanding benefits they bring humanity.

Abstract Background The most commonly occurring cancer mutations, including oncogenes such as MYC, Ras and PIK3C, are found in signal transductions pathways feeding into the translational machinery. A broad range of translation initiation factors are also commonly found to be either amplified or mis-regulated in tumours, including eIF4E (elongation initiation factor 4E). eIF4E is a subunit of the eIF4F protein initiation complex and required for its recruitment. Here we measure the formation of the eIF4F complex through interactions of eIF4E and eIF4G subunits, and the effect of oncogenic signalling pathways on complex formation. Results We developed a protein fragment complementation (PCA) assay that can accurately measure the status of the eIF4E-eIF4G interaction in cells and quantify the signalling flux through the RAS/ERK and PI3K/AKT pathways regulating eIF4F assembly. Complex disruption induced by inhibition of either pathway was shown to be a function of the phosphorylation status of 4EBP1, a key mediator of eIF4F assembly that interacts directly with eIF4E, confirming 4EBP1’s ability to integrate multiple signals affecting cap-dependent translation. Maximal measured disruption of the eIF4F complex occurred under combined mTORC1 and mTORC2 inhibition, whilst combined inhibition of both RAS/ERK and PI3K/AKT pathways in parallel resulted in greater inhibition of eIF4F formation than individually. v-Myc-mediated resistance to dual mTORC/PI3K inhibition was also principally demonstrated to depend on the lack of competent 4EBP1 available in the cell to bind eIF4E. Conclusions We show that 4EBP1 is a critical regulator of the mitogen responsive RAS/ERK and PI3K/AKT pathways and a key transducer of resistance mechanisms that affect small molecule inhibition of these pathways, principally by attenuating their effects on cap-dependent translation. These findings highlight the importance of highly efficacious direct inhibitors of eIF4E and eIF4F assembly, which could potentially target a wide spectrum of tumours containing differing mutations that effect these pathways and which confer chemo-resistance.

Summary: The development and uptake of citizen science and artificial intelligence (AI) techniques for ecological monitoring is increasing rapidly. Citizen science and AI allow scientists to create and process larger volumes of data than possible with conventional methods. However, managers of large ecological monitoring projects have little guidance on whether citizen science, AI, or both, best suit their resource capacity and objectives. To highlight the benefits of integrating the two techniques and guide future implementation by managers, we explore the opportunities, challenges, and complementarities of using citizen science and AI for ecological monitoring. We identify project attributes to consider when implementing these techniques and suggest that financial resources, engagement, participant training, technical expertise, and subject charisma and identification are important project considerations. Ultimately, we highlight that integration can supercharge outcomes for ecological monitoring, enhancing cost-efficiency, accuracy, and multi-sector engagement. The Bigger Picture: Citizen science and artificial intelligence (AI) are often used in isolation for ecological monitoring, but their integration likely has emergent benefits for management and scientific inquiry. We explore the complementarity of citizen science and AI for ecological monitoring, highlighting key opportunities and challenges. We show that strategic integration of citizen science and AI can improve outcomes for conservation activities. For example, coupling the public engagement benefits of citizen science with the advanced analytical capabilities of AI can increase multi-stakeholder accord on issues of public and scientific interest. Furthermore, both techniques speed up data collection and processing compared with conventional scientific techniques, suggesting that their integration can fast-track monitoring and conservation actions. We present key project attributes that will assist project managers in prioritizing the resources needed to implement citizen science, AI, or preferably both.

Abstract The growing demand for freshwater resources has led to dam construction and water diversions in a majority of the world's large rivers. With an increasing demand for freshwater, trade‐offs between water allocations and the preservation of ecological connections between terrestrial and marine ecosystems are inevitable. The ecological links formed by rivers flowing into the ocean benefit many commercially fished species. The degree to which different species and the livelihoods of fishers are negatively impacted by changes in river flows due to water extraction or diversion is important for management across terrestrial and marine boundaries. Our objective was to predict how changes in freshwater flows from three wet–dry tropical rivers in northern Australia, that is, the Mitchell, Gilbert, and Flinders rivers, affect the commercial banana prawn (Penaeus merguiensis) catch. We used a novel spatiotemporal Bayesian approach to model the effects of river flows and key climate drivers on banana prawn catch. We then predicted how the loss of flow due to water extraction or diversion affected prawn catch. Our analyses of three water development scenarios found that catch was most impacted by water extraction during low flows. The impact of water extraction was greatest for a scenario with dams on the Mitchell River, where we predicted catch would decline by 53% during a year with low flow. Overall, our results imply that maintenance of low‐level flows is a crucial requirement for sustained fishery yields. We suggest that water managers must balance agricultural demand for water during drier years against the impact of water extraction on prawn fisheries during low‐flow years. Protecting low‐level flows during drier years is a priority for maintaining terrestrial–marine linkages for adjacent marine fisheries.

Aquatic ecologists routinely count animals to provide critical information for conservation and management. Increased accessibility to underwater recording equipment such as action cameras and unmanned underwater devices has allowed footage to be captured efficiently and safely, without the logistical difficulties manual data collection often presents. It has, however, led to immense volumes of data being collected that require manual processing and thus significant time, labor, and money. The use of deep learning to automate image processing has substantial benefits but has rarely been adopted within the field of aquatic ecology. To test its efficacy and utility, we compared the accuracy and speed of deep learning techniques against human counterparts for quantifying fish abundance in underwater images and video footage. We collected footage of fish assemblages in seagrass meadows in Queensland, Australia. We produced three models using an object detection framework to detect the target species, an ecologically important fish, luderick (Girella tricuspidata). Our models were trained on three randomized 80:20 ratios of training:validation datasets from a total of 6,080 annotations. The computer accurately determined abundance from videos with high performance using unseen footage from the same estuary as the training data (F1 = 92.4%, mAP50 = 92.5%) and from novel footage collected from a different estuary (F1 = 92.3%, mAP50 = 93.4%). The computer’s performance in determining abundance was 7.1% better than human marine experts and 13.4% better than citizen scientists in single image test datasets, and 1.5 and 7.8% higher in video datasets, respectively. We show that deep learning can be a more accurate tool than humans at determining abundance and that results are consistent and transferable across survey locations. Deep learning methods provide a faster, cheaper, and more accurate alternative to manual data analysis methods currently used to monitor and assess animal abundance and have much to offer the field of aquatic ecology.

Abstract Structural habitat complexity is a fundamental attribute influencing ecological food webs. Simplification of complex habitats occurs due to both natural and anthropogenic pressures that can alter productivity of food webs. Relationships between food web structure and habitat complexity may be influenced by multiple mechanisms, and untangling these can be challenging. We investigated whether (1) size spectra vary across a gradient of habitat complexity in seagrass meadows and (2) structural complexity changes the importance of different primary producers supporting the food web (determined using stable isotope analysis) in the Great Barrier Reef World Heritage Area. We found that moderately complex meadows had much steeper size spectra slopes, caused by a higher abundance of smaller animals and fewer larger animals, while meadows on either end of the complexity scale (low and a single meadow with very high complexity) had shallower slopes, indicative of a more balanced distribution of animal sizes across the spectrum. We also found that the importance of epiphytic algae as a food source was high in most meadows, despite the increase in seagrass surface area on which epiphytes could grow. The consistent importance of epiphytic algae suggests that the changes in the availability of different potential food sources did not affect food web structure. Our findings indicate that food web structure may change with variations in structural complexity because of changes in the abundance of smaller and/or larger animals. Food web structure and food sources are important determinants of the dynamic stability of food webs. Size spectra analysis is already used as a monitoring tool for assessing populations of key fisheries species in commercial fishing operations, and thus, we recommend using size spectra as a proxy for assessing the structure of the food webs in different types of seagrass meadows. Size spectra may be a useful indicator of how different meadows provide for ecosystem services such as fisheries.

As longwall tailgate support technologies continue to be developed and improved, strata worldwide has worked to design and manufacture a revised Propsetter to provide additional support capacity and enable applications at greater mine heights. The support is targeted for tailgate applications, however, it can also be utilized for roof support in various types of mining environments. This paper describes the design changes for enhancing the support capacity of the Propsetter to more closely align its performance with other support technologies currently being employed in longwall tailgates. Balancing changes to each design aspect of the Propsetter was key to successfully improving the roof support’s load capacity while maintaining controlled deformation. Increasing the load capacity would allow a mine to improve support efficiency with a smaller, less intrusive support compared to support alternatives and reduce the cost per foot of supported entry. The target support capacity was 711–890 kN at 50 mm of deformation to more closely match the performance of competing tailgate supports such as the 610 mm-diameter CAN®. To achieve this goal, changes were made to all design aspects of the Propsetter: diameter, wedge cut depth and angle, pod size, and confinement rings. Test results from the NIOSH Mine Roof Simulator verified that these design changes were successful in increasing the capacity of the Propsetter to the 711–890 kN range at fifty mms of deformation. Keywords: Propsetter, Can, Longwall mining, Tailgate roof support, Controlled deformation

Abstract Coastal ecosystems can be degraded by poor water quality. Tracing the causes of poor water quality back to land-use change is necessary to target catchment management for coastal zone management. However, existing models for tracing the sources of pollution require extensive data-sets which are not available for many of the world’s coral reef regions that may have severe water quality issues. Here we develop a hierarchical Bayesian model that uses freely available satellite data to infer the connection between land-uses in catchments and water clarity in coastal oceans. We apply the model to estimate the influence of land-use change on water clarity in Fiji. We tested the model’s predictions against underwater surveys, finding that predictions of poor water quality are consistent with observations of high siltation and low coverage of sediment-sensitive coral genera. The model thus provides a means to link land-use change to declines in coastal water quality.

Seascapes are typically comprised of multiple components that are functionally linked by the movement of organisms and fluxes of matter. Changes to the number and spatial arrangement of these linkages affect biological connectivity that, in turn, can alter ecological functions. Herbivory is one such function, pivotal in controlling excessive algal growth when systems become disturbed. Here, we used microcosm experiments to test how the change to connectivity affects herbivory under different levels of disturbance. We applied network theory to measure types of connectivity at different scales (patch and whole system) and quantified herbivory by a crustacean mesograzer exposed to excess algae, mimicking pulse and press disturbances. We demonstrate that greater connectivity significantly enhances herbivory in Clibanarius virescens: Both the number of linkages and their spatial arrangement interact to shape the response of herbivory in systems to disturbance. Our findings highlight the value of controlled experiments for advancing theories about the potential effects of connectivity on important ecological functions, such as herbivory, and justify further investigation to measure how connectivity might affect the resilience of ecosystems. We posit that the variation in the type, and scale, of spatial linkages might have profound consequences for managing the capacity of ecosystems to respond to disturbance.

Stapled peptides targeting the interaction between p53 and its negative regulators MDM2 and MDM4 have exhibited great potential as anti-cancer drugs, albeit with room for improvement in formulation and tumor specificity. Lipid bilayer disks (lipodisks) have emerged as promising drug nanocarriers and can by attachment of targeting moieties be directed selectively towards tumor cells. Tumor-targeted delivery of stapled peptides by use of lipodisks may therefore increase the uptake in the tumors and limit toxicity in healthy tissue. Here, we utilized epidermal growth factor receptor (EGFR)-targeted lipodisks to deliver p53-activating stapled peptide VIP116 to EGFR-expressing tumor cells. We demonstrate that VIP116 can be stably formulated in lipodisks (maximum peptide/lipid molar ratio 0.11). In vitro cell studies verify specific binding of EGF-decorated lipodisks to tumor cells and confirm that targeted delivery of VIP116 significantly decreases tumor cell viability.

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.