The mechanism of covalent inhibition of LAR phosphatase by illudalic acid.

Leukocyte antigen-related (LAR) phosphatase is a receptor-type protein tyrosine phosphatase involved in cellular signaling and associated with human disease including cancer and metabolic disorders. Selective inhibition of LAR phosphatase activity by well characterized and well validated small molecules would provide key insights into the roles of LAR phosphatase in health and disease, but identifying selective inhibitors of LAR phosphatase activity has been challenging. Recently, we described potent and selective inhibition of LAR phosphatase activity by the fungal natural product illudalic acid. Here we provide a detailed biochemical characterization of the adduct formed between LAR phosphatase and illudalic acid. A mass spectrometric analysis indicates that two cysteine residues are covalently labeled by illudalic acid and a related analog. Mutational analysis supports the hypothesis that inhibition of LAR phosphatase activity is due primarily to the adduct with the catalytic cysteine residue. A computational study suggests potential interactions between the illudalic acid moiety and the enzyme active site. Taken together, these data offer novel insights into the mechanism of inhibition of LAR phosphatase activity by illudalic acid.

van der Waals Parameter Scanning with Amber Nucleic Acid Force Fields: Revisiting Means to Better Capture the RNA/DNA Structure through MD.

Molecular dynamics simulations can be used in combination with experimental techniques to uncover the intricacies of biomolecular structure, dynamics, and the resulting interactions. However, many noncanonical nucleic acid structures have proven to be challenging to replicate in accurate agreement with experimental data, often attributed to known force field deficiencies. A common force field criticism is the handling of van der Waals (vdW) parameters, which have not been updated since the regular use of Ewald's methods became routine. This work dives into the effects of minute vdW radii shifts on RNA tetranucleotide, B-DNA, and Z-DNA model systems described by commonly used Amber force fields. Using multidimensional replica exchange molecular dynamics (M-REMD), the GACC RNA tetranucleotide demonstrated changes in the structural distribution between the NMR minor and anomalous structure populations based on the O2' vdW radii scanning. However, no significant change in the NMR Major conformation population was observed. There were minimal changes in the B-DNA structure but there were more substantial improvements in Z-DNA structural descriptions, specifically with the Tumuc1 force field. This occurred with both LJbb vdW radii adjustments and incorporation of the CUFIX nonbonded parameter modifications. Though the limited vdW modifications tested did not provide a universal fix to the challenge of simulating the various known nucleic acid structures, they do provide direction and a greater understanding for future force field development efforts.

Computational Modeling of Stapled Coiled-Coil Inhibitors Against Bcr-Abl: Toward a Treatment Strategy for CML.

The chimeric oncoprotein Bcr-Abl is the causative agent of virtually all chronic myeloid leukemias (CML) and a subset of acute lymphoblastic leukemias (ALL). As a result of the so-called Philadelphia Chromosome translocation t(9;22), Bcr-Abl manifests as a constitutively active tyrosine kinase which promotes leukemogenesis by activation of cell cycle signaling pathways. Constitutive and oncogenic activation is mediated by an N-terminal coiled-coil oligomerization domain in Bcr (Bcr-CC), presenting a therapeutic target for inhibition of Bcr-Abl activity toward the treatment of Bcr-Abl+ leukemias. Previously, we demonstrated that a rationally designed Bcr-CC mutant, CCmut3, exerts a dominant negative effect upon Bcr-Abl activity by preferential oligomerization with Bcr-CC. Moreover, we have shown conjugation to a leukemia-specific cell-penetrating peptide (CPP-CCmut3) improves intracellular delivery and activity. However, our full-length CPP-CCmut3 construct (81 aa) is encumbered by an intrinsically high degree of conformational variability and susceptibility to proteolytic degradation, relative to traditional small molecule therapeutics. Here, we iterate a new generation of our inhibitor against Bcr-CC mediated Bcr-Abl assembly that is designed to address these constraints through incorporation of all-hydrocarbon staples spanning i, i + 7 positions in helix α2 (CPP-CCmut3-st). We utilize computational modeling and biomolecular simulation to design and characterize single and double staple candidates in silico, evaluating binding energetics and building upon our seminal work modeling single hydrocarbon staples when applied to a truncated Bcr-CC sequence. This strategy enables us to efficiently build, characterize, and screen lead single/double stapled CPP-CCmut3-st candidates for experimental studies and validation in vitro and in vivo. In addition to full-length CPP-CCmut, we model a truncated system characterized by deletion of helix α1 and the flexible-loop linker, which are known to impart high conformational variability. To study the impact of the N-terminal cyclic CPP toward model stability and inhibitor activity, we also model the full-length and truncated systems without CPP, with cyclized CPP, and with linear CPP, for a total of six systems which comprise our library. From this library, we present lead stapled peptide candidates to be synthesized and evaluated experimentally as our next-generation inhibitors against Bcr-Abl.

Assessment of A- to B- DNA Transitions Utilizing the Drude Polarizable Force Field.

In addition to the well-characterized B-form of DNA, duplex DNA can adopt various conformations, such as A or Z-DNA. Though less common, these structures can be induced biologically through protein or ligand interactions or experimentally with niche environmental conditions, such as high salt concentrations or in mixed water-ethanol. Reproducing these alternate structures through molecular dynamics simulations in recent years has been quite challenging with the currently available force fields, simulation techniques, and time scales. In this study, the Drude polarizable force field is tested for its ability to facilitate transitions between A-DNA and B-DNA or maintain A-DNA. Though transitions away from B-DNA were observed in high concentrations of ethanol, the resulting structures had hybrid properties taken from both B-DNA and A-DNA structures. This was also true for A-DNA in ethanol, which lost some of the A-DNA properties that it was expected to maintain. When B-DNA was tested in high salt environments, the resulting B-DNA structures showed no distinguishable differences with the increasing salt concentrations tested. These results with the Drude FF and recent results with additive force fields suggest that at present the current additive and polarizable force fields do not facilitate a complete transition between B- to A-DNA conformations under the conditions simulated. At present, the Drude FF favors A-B DNA hybrid structures when simulated in nonphysiological conditions.

AmberTools.

AmberTools is a free and open-source collection of programs used to set up, run, and analyze molecular simulations. The newer features contained within AmberTools23 are briefly described in this Application note.

Assessing the Current State of Amber Force Field Modifications for DNA─2023 Edition.

Advances in molecular dynamics (MD) software alongside enhanced computational power and hardware have allowed for MD simulations to significantly expand our knowledge of biomolecular structure, dynamics, and interactions. Furthermore, it has allowed for the extension of conformational sampling times from nanoseconds to the microsecond level and beyond. This has not only made convergence of conformational ensembles through comprehensive sampling possible but consequently exposed deficiencies and allowed the community to overcome limitations in the available force fields. The reproducibility and accuracy of the force fields are imperative in order to produce biologically relevant data. The Amber nucleic acid force fields have been used widely since the mid-1980s, and improvement of these force fields has been a community effort with several artifacts revealed, corrected, and reevaluated by various research groups. Here, we focus on the Amber force fields for use with double-stranded DNA and present the assessment of two recently developed force field parameter sets (OL21 and Tumuc1). Extensive MD simulations were performed with six test systems and two different water models. We observe the improvement of OL21 and Tumuc1 compared to previous generations of the Amber DNA force. We did not detect any significant improvement in the performance of Tumuc1 compared to OL21 despite the reparameterization of bonded force field terms in the former; however, we did note discrepancies in Tumuc1 when modeling Z-DNA sequences.

Benchmarking the Drude Polarizable Force Field Using the r(GACC) Tetranucleotide.

Polarizable force fields, in particular, the Drude polarizable force field (Drude FF), may hold the key to more accurately modeling biomolecules with molecular dynamics simulations by explicitly accounting for atomic polarizability. Previous work has shown promising results in simulating duplex nucleic acids and protein structures with excellent agreement with experimental values. However, benchmarking the Drude polarizable force field with highly flexible, single-stranded structures has yet to be achieved. In this work, the r(GACC) tetranucleotide is simulated over a multimicrosecond time scale, starting with various different initial conformations. Despite the starting conformation, including starting from the expected dominant A-form major conformation, the experimental structural distribution is not matched. In fact, the major NMR conformation is never resampled. Instead, the r(GACC) tetranucleotide becomes stabilized in anomalous structures that are inconsistent with the NMR data and that favor base-pairing and electrostatic interactions over base stacking. These structures are maintained for lengthy time scales (>1 µs) themselves, suggesting a misbalance of forces in the Drude polarizable force field itself. This model system is suggestive of the fact that currently the Drude polarizable force field does not appear to produce the sensitive balance of forces required to accurately model other single-stranded or noncanonical RNA structures.

Structures and Dynamics of DNA Mini-Dumbbells Are Force Field Dependent.

Flexible nucleic acid structures can be challenging to accurately resolve with currently available experimental structural determination techniques. As an alternative, molecular dynamics (MD) simulations can provide a window into understanding the unique dynamics and population distributions of these biomolecules. Previously, molecular dynamics simulations of noncanonical (non-duplex) nucleic acids have proven difficult to accurately model. With a new influx of improved nucleic acid force fields, achieving an in-depth understanding of the dynamics of flexible nucleic acid structures may be achievable. In this project, currently available nucleic acid force fields are evaluated using a flexible yet stable model system: the DNA mini-dumbbell. Prior to MD simulations, nuclear magnetic resonance (NMR) re-refinement was accomplished using improved refinement techniques in explicit solvent to yield DNA mini-dumbbell structures with better agreement between the newly determined PDB snapshots, with the NMR data itself, as well as the unrestrained simulation data. Starting from newly determined structures, a total aggregate of over 800 µs of production data between 2 DNA mini-dumbbell sequences and 8 force fields was collected to compare to these newly refined structures. The force fields tested spanned from traditional Amber force fields: bsc0, bsc1, OL15, and OL21 to Charmm force fields: Charmm36 and the Drude polarizable force field, as well as force fields from independent developers: Tumuc1 and CuFix/NBFix. The results indicated slight variations not only between the different force fields but also between the sequences as well. Given our previous experiences with high populations of potentially anomalous structures in RNA UUCG tetraloops and in various tetranucleotides, we expected the mini-dumbbell system to be challenging to accurately model. Surprisingly, many of the recently developed force fields generated structures in good agreement with experiments. Yet, each of the force fields provided a different distribution of potentially anomalous structures.

Systematic Investigation of Tether Length and Phosphorus Configuration in Backbone Constrained Macrocyclic Nucleic Acids to Modulate Binding Kinetics for RNA.

We recently described a chemical strategy to pre-organize a trinucleotide subunit in a conformation suitable for Watson-Crick base pairing for modulating the binding kinetics of single-stranded oligonucleotides (ONs) using bis-phosphonate esters bridging hydrocarbon tethers to provide 11- and 15-membered macrocyclic analogues. In this manuscript, we describe the synthesis of all eight P-stereoisomers of macrocyclic 12-, 13-, 14-, and 16-membered hydrocarbon-bridged nucleotide trimers, their incorporation into ONs, and biophysical characterization of the modified ONs. The size of the macrocyclic tether and configuration at phosphorus had profound effects on hybridization kinetics. ONs containing 12- and 13-membered rings exhibited faster on-rates (up to 5-fold) and off-rates (up to 161-fold). In contrast, ONs using the larger ring size macrocycles generally exhibited smaller changes in binding kinetics relative to unmodified DNA. Interestingly, several of the analogues retained significant binding affinity for RNA based on their dissociation constants, despite being modestly destabilizing in the thermal denaturation experiments, highlighting the potential utility of measuring dissociation constants versus duplex thermal stability when evaluating novel nucleic acid analogues. Overall, our results provide additional insights into the ability of backbone-constrained macrocyclic nucleic acid analogues to modulate hybridization kinetics of modified ONs with RNA.

Transient Hoogsteen Base Pairs Observed in Unbiased Molecular Dynamics Simulations of DNA.

Duplex DNA is modeled as canonical B-DNA displaying the characteristic Watson-Crick base pairs. A less common and short-lived pairing of the nucleobases is the Hoogsteen (HG) conformation. The low population of the HG base pairs (<1%) necessitates extended sampling times in order to analyze through unbiased molecular dynamics (MD) simulations. We have discovered that with extended sampling times using multiple independent copies of an 18-mer sequence, the MD trajectories reproduce the expected and transient HG base pairing. Consistent with experiment, the percentage of the HG events are within the range of ∼0.1-1.0% over the combined aggregate sampling time of more than 3.6 ms. We present the reliability of the current AMBER set of nucleic acid force fields and tools to accurately simulate naturally occurring base-pairing and opening events without any bias or restraints. The mechanism consists of base pair fraying, flipping of the purine, and reformation with HG base pairs.

Riboflavin Stabilizes Abasic, Oxidized G-Quadruplex Structures.

The G-quadruplex is a noncanonical fold of DNA commonly found at telomeres and within gene promoter regions of the genome. These guanine-rich sequences are highly susceptible to damages such as base oxidation and depurination, leading to abasic sites. In the present work, we address whether a vacancy, such as an abasic site, in a G-quadruplex serves as a specific ligand recognition site. When the G-tetrad is all guanines, the vacant (abasic) site is recognized and bound by free guanine nucleobase. However, we aim to understand whether the preference for a specific ligand recognition changes with the presence of a guanine oxidation product 8-oxo-7,8-dihydroguanine (OG) adjacent to the vacancy in the tetrad. Using molecular dynamics simulation, circular dichroism, and nuclear magnetic resonance, we examined the ability for riboflavin to stabilize abasic site-containing G-quadruplex structures. Through structural and free energy binding analysis, we observe riboflavin's ability to stabilize an abasic site-containing G-quadruplex only in the presence of an adjacent OG-modified base. Further, when compared to simulation with the vacancy filled by free guanine, we observe that the free guanine nucleobase is pushed outside of the tetrad by OG to interact with other parts of the structure, including loop residues. These results support the preference of riboflavin over free guanine to fill an OG-adjacent G-quadruplex abasic vacancy.

Backbone Hydrocarbon-Constrained Nucleic Acids Modulate Hybridization Kinetics for RNA.

The binding affinity of therapeutic oligonucleotides (ONs) for their cognate RNA is determined by the rates of association (ka) and dissociation (kd). Single-stranded ONs are highly flexible and can adopt multiple conformations in solution, some of which may not be conducive for hybridization. We investigated if restricting rotation around the sugar-phosphate backbone, by tethering two adjacent backbone phosphonate esters using hydrocarbon bridges, can modulate hybridization kinetics of the modified ONs for complementary RNA. Given the large number of possible analogues with different tether lengths and configurations at the phosphorus atoms, we employed molecular dynamic simulations to optimize the size of the hydrocarbon bridge to guide the synthetic efforts. The backbone-constrained nucleotide trimers with stereodefined configurations at the contiguous backbone phosphorus atoms were assembled using a ring-closing metathesis reaction, then incorporated into oligonucleotides by an in situ synthesis of the phosphoramidites followed by coupling to solid supports. Evaluation of the modified oligonucleotides revealed that 15-membered macrocyclic-constrained analogues displayed similar or slightly improved on-rates but significantly increased off-rates compared to unmodified DNA ONs, resulting in reduced duplex stability. In contrast, LNA ONs with conformationally preorganized furanose rings showed similar on-rates to DNA ONs but very slow off-rates, resulting in net improvement in duplex stability. Furthermore, the experimental data generally supported the molecular dynamics simulation results, suggesting that this strategy can be used as a predictive tool for designing the next generation of constrained backbone ON analogues with improved hybridization properties.

Peptoid Residues Make Diverse, Hyperstable Collagen Triple-Helices.

As the only ribosomally encoded N-substituted amino acid, proline promotes distinct secondary protein structures. The high proline content in collagen, the most abundant protein in the human body, is crucial to forming its hallmark structure: the triple-helix. For over five decades, proline has been considered compulsory for synthetic designs aimed at recapitulating collagen's structure and properties. Here we describe that N-substituted glycines (N-glys), also known as peptoid residues, exhibit a general triple-helical propensity similar to or greater than proline, enabling synthesis of stable triple-helical collagen mimetic peptides (CMPs) with unprecedented side chain diversity. Supported by atomic-resolution crystal structures as well as circular dichroism and computational characterizations spanning over 30 N-gly-containing CMPs, we discovered that N-glys stabilize the triple-helix primarily by sterically preorganizing individual chains into the polyproline-II helix. We demonstrated that N-glys with exotic side chains including a "click"-able alkyne and a photosensitive side chain enable CMPs for functional applications including the spatiotemporal control of cell adhesion and migration. The structural principles uncovered in this study open up opportunities for a new generation of collagen-mimetic therapeutics and materials.

Ethidium bromide interactions with DNA: an exploration of a classic DNA-ligand complex with unbiased molecular dynamics simulations.

Visualization of double stranded DNA in gels with the binding of the fluorescent dye ethidium bromide has been a basic experimental technique in any molecular biology laboratory for >40 years. The interaction between ethidium and double stranded DNA has been observed to be an intercalation between base pairs with strong experimental evidence. This presents a unique opportunity for computational chemistry and biomolecular simulation techniques to benchmark and assess their models in order to see if the theory can reproduce experiments and ultimately provide new insights. We present molecular dynamics simulations of the interaction of ethidium with two different double stranded DNA models. The first model system is the classic sequence d(CGCGAATTCGCG)2 also known as the Drew-Dickerson dodecamer. We found that the ethidium ligand binds mainly stacked on, or intercalated between, the terminal base pairs of the DNA with little to no interaction with the inner base pairs. As the intercalation at the terminal CpG steps is relatively rapid, the resultant DNA unwinding, rigidification, and increased stability of the internal base pair steps inhibits further intercalation. In order to reduce these interactions and to provide a larger groove space, a second 18-mer DNA duplex system with the sequence d(GCATGAACGAACGAACGC) was tested. We computed molecular dynamics simulations for 20 independent replicas with this sequence, each with ∼27 µs of sampling time. Results show several spontaneous intercalation and base-pair eversion events that are consistent with experimental observations. The present work suggests that extended MD simulations with modern DNA force fields and optimized simulation codes are allowing the ability to reproduce unbiased intercalation events that we were not able to previously reach due to limits in computing power and the lack of extensively tested force fields and analysis tools.

Ancillary Ligand in Ternary CuII Complexes Guides Binding Selectivity toward Minor-Groove DNA.

Copper-containing compounds known as Casiopeínas are biologically active molecules which show promising antineoplastic effects against several cancer types. Two possible hypotheses regarding the mode of action of the Casiopeínas have emerged from the experimental evidence: the generation of reactive oxygen species or the ability of the compounds to bind and interact with nucleic acids. Using robust molecular dynamics simulations, we investigate the interaction of four different Casiopeínas with the DNA duplex d(GCACGAACGAACGAACGC). The studied copper complexes contain either 4-7- or 5-6-substituted dimethyl phenanthroline as the primary ligand and either glycinate or acetylacetonate as the secondary ligand. For statistical significance and to reduce bias in the simulations, four molecules of each copper compound were manually placed at a distance of 10 Å away from the DNA and 20 independent molecular dynamics simulations were performed, each reaching at least 30 µs. This time scale allows us to reproduce expected DNA terminal base-pair fraying and also to observe intercalation/base-pair eversion events generated by the compounds interacting with DNA. The results reveal that the secondary ligand is the guide toward the mode of binding between the copper complex and DNA in which glycinate prefers minor-groove binding and acetylacetonate produces base-pair eversion and intercalation. The CuII complexes containing glycinate interact within the DNA minor groove which are stabilized principally by the hydrogen bonds formed between the amino group of the aminoacidate moiety, whereas the compounds with the acetylacetonate do not present a stable network of hydrogen bonds and the ligand interactions enhance DNA breathing dynamics that result in base-pair eversion.

Exploring potentially alternative non-canonical DNA duplex structures through simulation.

Hopkins proposed an alternative and chirally distinct family of double-stranded DNA (dsDNA) models that have antiparallel chains with 5'→3' senses opposite to those of the right-handed Watson-Crick (WC) family. Termed configuration II, this family of dsDNA models contains both right-handed (II-R) and left-handed (II-L) forms, with Z-DNA as an example of the latter. Relative interstrand binding energies for six DNA duplex models, two each of configuration I-R (standard WC canonical B-DNA), II-R, and II-L for the duplex d(CGCGAATTCGCG), have been estimated under identical conditions using MM-PBSA analysis from molecular dynamics trajectories using three different AMBER force fields. These simulations support the stereo chemical soundness of configuration II dsDNA forms. Recent force fields (Barcelona Supercomputing Center [BSC] [bsc1] and Olomouc 2015 [OL15]) successfully render stable II-L structures, whereas the previous force field, bsc0, generated stable II-R structures, although with an energy difference between II-R and II-L of ∼30 kcal/mol. Communicated by Ramaswamy H. Sarma.

Lessons learned in atomistic simulation of double-stranded DNA: Solvation and salt concerns [Article v1.0].

Nucleic acids are highly charged macromolecules sensitive to their surroundings of water, salt, and other biomolecules. Molecular dynamics simulations with accurate biomolecular force fields provide a detailed atomistic view into DNA and RNA that has been useful to study the structure and dynamics of these molecules and their biological relevance. In this work we study the Drew-Dickerson dodecamer duplex with the sequence d(GCGCAATTGCGC)2 in three different salt concentrations and using different monvalent salt types to detect possible structural influence. Overall, the DNA shows no major structural changes regardless of amount or type of monovalent ions used. Our results show that only at very high salt conditions (5M) is a small structural effect observed in the DNA duplex, which mainly consist of narrowing of the grooves due to increased residence of ions. We also present the importance of sampling time to achieve a converged ensemble, which is of major relevance in any simulation to avoid biased or non-meaningful results.

Parallelization of CPPTRAJ enables large scale analysis of molecular dynamics trajectory data.

Advances in biomolecular simulation methods and access to large scale computer resources have led to a massive increase in the amount of data generated. The key enablers have been optimization and parallelization of the simulation codes. However, much of the software used to analyze trajectory data from these simulations is still run in serial, or in some cases many threads via shared memory. Here, we describe the addition of multiple levels of parallel trajectory processing to the molecular dynamics simulation analysis software CPPTRAJ. In addition to the existing OpenMP shared-memory parallelism, CPPTRAJ now has two additional levels of message passing (MPI) parallelism involving both across-trajectory processing and across-ensemble processing. All three levels of parallelism can be simultaneously active, leading to significant speed ups in data analysis of large datasets on the NCSA Blue Waters supercomputer by better leveraging the many available nodes and its parallel file system. © 2018 Wiley Periodicals, Inc.

Defining a conformational ensemble that directs activation of PPARγ.

The nuclear receptor ligand-binding domain (LBD) is a highly dynamic entity. Crystal structures have defined multiple low-energy LBD structural conformations of the activation function-2 (AF-2) co-regulator-binding surface, yet it remains unclear how ligand binding influences the number and population of conformations within the AF-2 structural ensemble. Here, we present a nuclear receptor co-regulator-binding surface structural ensemble in solution, viewed through the lens of fluorine-19 (19F) nuclear magnetic resonance (NMR) and molecular simulations, and the response of this ensemble to ligands, co-regulator peptides and heterodimerization. We correlate the composition of this ensemble with function in peroxisome proliferator-activated receptor-γ (PPARγ) utilizing ligands of diverse efficacy in co-regulator recruitment. While the co-regulator surface of apo PPARγ and partial-agonist-bound PPARγ is characterized by multiple thermodynamically accessible conformations, the full and inverse-agonist-bound PPARγ co-regulator surface is restricted to a few conformations which favor coactivator or corepressor binding, respectively.

Computational Modeling of Stapled Peptides toward a Treatment Strategy for CML and Broader Implications in the Design of Lengthy Peptide Therapeutics.

The oncogenic gene product Bcr-Abl is the principal cause of chronic myeloid leukemia, and although several therapies exist to curb the aberrant kinase activity of Bcr-Abl through targeting of the Abl kinase domain, these therapies are rendered ineffective by frequent mutations in the corresponding gene. It has been demonstrated that a designed protein, known as CCmut3, is able to produce a dominant negative inactivating effect on Bcr-Abl kinase by preferentially oligomerizing with the N-terminal coiled-coil oligomerization domain of Bcr-Abl (Bcr-CC) to effectively reduce the oncogenic potential of Bcr-Abl. However, the sheer length of the CCmut3 peptide introduces a high degree of conformational variability and opportunity for targeting by intracellular proteolytic mechanisms. Here, we have examined the effects of introducing one or two molecular staples, or cross-links, spanning i, i + 7 backbone residues of the CCmut3 construct, which have been suggested to reinforce α-helical conformation, enhance cellular internalization, and increase resistance to proteolytic degradation, leading to enhanced pharmacokinetic properties. The importance of optimizing staple location along a highly tuned biological construct such as CCmut3 has been widely emphasized and, as such, we have employed in silico techniques to swiftly build, relax, and characterize a large number of candidates. This approach effectively allowed exploring each and every possible staple location along the peptide backbone so that every possible candidate is considered. Although many of the stapled candidate peptides displayed enhanced binding characteristics for Bcr-CC and improved conformational stability in the (Bcr-CC) bound form, simulations of the stapled peptides in the unbound form revealed widespread conformational variability among stapled candidates dependent on staple type and location, implicating the molecular replacement of helix-stabilizing residues with staple-containing residues in disrupting the native α-helical conformation of CCmut3, further highlighting a need for careful optimization of the CCmut3 construct. A candidate set has been assembled, which retains the native backbone α-helical integrity in both the bound and unbound forms while providing enhanced binding affinity for the Bcr-CC target, as research disseminated in this manuscript is intended to guide the development of a next-generation CCmut3 inhibitor peptide in an experimental setting.