Design-rules for stapled peptides with in vivo activity and their application to Mdm2/X antagonists.

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.

Liposome Click Membrane Permeability Assay for Identifying Permeable Peptides.

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

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

Macrocyclic peptides have the potential to address intracellular protein-protein interactions (PPIs) of high value therapeutic targets that have proven largely intractable to small molecules. Here, we report broadly applicable lessons for applying this modality to intracellular targets and specifically for advancing chemical matter to address KRAS, a protein that represents the most common oncogene in human lung, colorectal and pancreatic cancers yet is one of the most challenging targets in human disease. Specifically, we focused on KRpep-2d, an arginine-rich KRAS-binding peptide with a disulfide-mediated macrocyclic linkage and a protease-sensitive backbone. These latter redox and proteolytic labilities obviated cellular activity. Extensive structure-activity relationship studies involving macrocyclic linker replacement, stereochemical inversion, and backbone α-methylation, gave a peptide with on-target cellular activity. However, we uncovered an important generic insight - the arginine-dependent cell entry mechanism limited its therapeutic potential. In particular, we observed a strong correlation between net positive charge and histamine release in an ex vivo assay, thus making this series unsuitable for advancement due to the potentially fatal consequences of mast cell degranulation. This observation should signal to researchers that cationic-mediated cell entry - an approach that has yet to succeed in the clinic despite a long history of attempts - carries significant therapy-limiting safety liabilities. Nonetheless, the cell-active molecules identified here validate a unique inhibitory epitope on KRAS and thus provide valuable molecular templates for the development of therapeutics that are desperately needed to address KRAS-driven cancers - some of the most treatment-resistant human malignancies.

De-risking Drug Discovery of Intracellular Targeting Peptides: Screening Strategies to Eliminate False-Positive Hits.

Nonspecific promiscuous compounds can mislead researchers and waste significant resources. This phenomenon, though well-documented for small molecules, has not been widely explored for the peptide modality. Here we demonstrate that two purported peptide-based KRas inhibitors, SAH-SOS1 A and cyclorasin 9A5, exemplify false-positive molecules-in terms of both their binding affinities and cellular activities. Through multiple gold-standard biophysical techniques, we unambiguously show that both peptides lack specific binding to KRas and instead induce protein unfolding. Although these peptides inhibited cellular proliferation, the activities appeared to be off-target on the basis of a counterscreen with KRas-independent cell lines. We further demonstrate that their cellular activities are derived from membrane disruption. Accordingly, we propose that to de-risk false-positive molecules, orthogonal binding assays and cellular counterscreens are indispensable.

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.

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.

Incorporation of Putative Helix-Breaking Amino Acids in the Design of Novel Stapled Peptides: Exploring Biophysical and Cellular Permeability Properties.

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

Macrocyclic α helical peptide therapeutic modality: A perspective of learnings and challenges.

Macrocyclic α-helical peptides have emerged as a compelling new therapeutic modality to tackle targets confined to the intracellular compartment. Within the scope of hydrocarbon-stapling there has been significant progress to date, including the first stapled α-helical peptide to enter into clinical trials. The principal design concept of stapled α-helical peptides is to mimic a cognate (protein) ligand relative to binding its target via an α-helical interface. However, it was the proclivity of such stapled α-helical peptides to exhibit cell permeability and proteolytic stability that underscored their promise as unique macrocyclic peptide drugs for intracellular targets. This perspective highlights key learnings as well as challenges in basic research with respect to structure-based design, innovative chemistry, cell permeability and proteolytic stability that are essential to fulfill the promise of stapled α-helical peptide drug development.

Targeting YAP/TAZ-TEAD protein-protein interactions using fragment-based and computational modeling approaches.

The Hippo signaling pathway, which is implicated in the regulation of organ size, has emerged as a potential target for the development of cancer therapeutics. YAP, TAZ (transcription co-activators) and TEAD (transcription factor) are the downstream transcriptional machinery and effectors of the pathway. Formation of the YAP/TAZ-TEAD complex leads to transcription of growth-promoting genes. Conversely, disrupting the interactions of the complex decreases cell proliferation. Herein, we screened a 1000-member fragment library using Thermal Shift Assay and identified a hit fragment. We confirmed its binding at the YAP/TAZ-TEAD interface by X-ray crystallography, and showed that it occupies the same hydrophobic pocket as a conserved phenylalanine of YAP/TAZ. This hit fragment serves as a scaffold for the development of compounds that have the potential to disrupt YAP/TAZ-TEAD interactions. Structure-activity relationship studies and computational modeling were also carried out to identify more potent compounds that may bind at this validated druggable binding site.

Crystal structure of TAZ-TEAD complex reveals a distinct interaction mode from that of YAP-TEAD complex.

The Hippo pathway is a tumor suppressor pathway that is implicated in the regulation of organ size. The pathway has three components: the upstream regulatory factors, the kinase core, and the downstream transcriptional machinery, which consists of YAP, TAZ (transcription co-activators) and TEAD (transcription factor). Formation of YAP/TAZ-TEAD complexes leads to the transcription of growth-promoting genes. Herein, we report the crystal structure of TAZ-TEAD4 complex, which reveals two binding modes. The first is similar to the published YAP-TEAD structure. The second is a unique binding mode, whereby two molecules of TAZ bind to and bridge two molecules of TEAD4. We validated the latter using cross-linking and multi-angle light scattering. Using siRNA, we showed that TAZ knockdown leads to a decrease in TEAD4 dimerization. Lastly, results from luciferase assays, using YAP/TAZ transfected or knockdown cells, give support to the non-redundancy of YAP/TAZ co-activators in regulating gene expression in the Hippo pathway.

Structural basis of Ornithine Decarboxylase inactivation and accelerated degradation by polyamine sensor Antizyme1.

Ornithine decarboxylase (ODC) catalyzes the first and rate-limiting step of polyamine biosynthesis in humans. Polyamines are essential for cell proliferation and are implicated in cellular processes, ranging from DNA replication to apoptosis. Excessive accumulation of polyamines has a cytotoxic effect on cells and elevated level of ODC activity is associated with cancer development. To maintain normal cellular proliferation, regulation of polyamine synthesis is imposed by Antizyme1 (AZ1). The expression of AZ1 is induced by a ribosomal frameshifting mechanism in response to increased intracellular polyamines. AZ1 regulates polyamine homeostasis by inactivating ODC activity and enhancing its degradation. Here, we report the structure of human ODC in complex with N-terminally truncated AZ1 (cAZ1). The structure shows cAZ1 binding to ODC, which occludes the binding of a second molecule of ODC to form the active homodimer. Consequently, the substrate binding site is disrupted and ODC is inactivated. Structural comparison shows that the binding of cAZ1 to ODC causes a global conformational change of ODC and renders its C-terminal region flexible, therefore exposing this region for degradation by the 26S proteasome. Our structure provides the molecular basis for the inactivation of ODC by AZ1 and sheds light on how AZ1 promotes its degradation.

"Snapshots" of ispinesib-induced conformational changes in the mitotic kinesin Eg5.

Kinesins comprise a superfamily of molecular motors that drive a wide variety of cellular physiologies, from cytoplasmic transport to formation of the bipolar spindle in mitosis. These differing roles are reflected in corresponding polymorphisms in key kinesin structural elements. One of these is a unique loop and stem motif found in all kinesins and referred to as loop 5 (L5). This loop is longest in the mitotic kinesin Eg5 and is the target for a number of small molecule inhibitors, including ispinesib, which is being used in clinical trials in patients with cancer. In this study, we have used x-ray crystallography to identify a new structure of an Eg5-ispinesib complex and have combined this with transient state kinetics to identify a plausible sequence of conformational changes that occur in response to ispinesib binding. Our results demonstrate that ispinesib-induced structural changes in L5 from Eg5 lead to subsequent changes in the conformation of the switch II loop and helix and in the neck linker. We conclude that L5 in Eg5 simultaneously regulates the structure of both the ATP binding site and the motor's mechanical elements that generate force.

Optimized S-trityl-L-cysteine-based inhibitors of kinesin spindle protein with potent in vivo antitumor activity in lung cancer xenograft models.

The mitotic kinesin Eg5 is critical for the assembly of the mitotic spindle and is a promising chemotherapy target. Previously, we identified S-trityl-L-cysteine as a selective inhibitor of Eg5 and developed triphenylbutanamine analogues with improved potency, favorable drug-like properties, but moderate in vivo activity. We report here their further optimization to produce extremely potent inhibitors of Eg5 (K(i)(app) < 10 nM) with broad-spectrum activity against cancer cell lines comparable to the Phase II drug candidates ispinesib and SB-743921. They have good oral bioavailability and pharmacokinetics and induced complete tumor regression in nude mice explanted with lung cancer patient xenografts. Furthermore, they display fewer liabilities with CYP-metabolizing enzymes and hERG compared with ispinesib and SB-743921, which is important given the likely application of Eg5 inhibitors in combination therapies. We present the case for this preclinical series to be investigated in single and combination chemotherapies, especially targeting hematological malignancies.

Triphenylbutanamines: kinesin spindle protein inhibitors with in vivo antitumor activity.

The human mitotic kinesin Eg5 represents a novel mitotic spindle target for cancer chemotherapy. We previously identified S-trityl-l-cysteine (STLC) and related analogues as selective potent inhibitors of Eg5. We herein report on the development of a series of 4,4,4-triphenylbutan-1-amine inhibitors derived from the STLC scaffold. This new generation systematically improves on potency: the most potent C-trityl analogues exhibit K(i)(app) ≤ 10 nM and GI(50) ≈ 50 nM, comparable to results from the phase II clinical benchmark ispinesib. Crystallographic studies reveal that they adopt the same overall binding configuration as S-trityl analogues at an allosteric site formed by loop L5 of Eg5. Evaluation of their druglike properties reveals favorable profiles for future development and, in the clinical candidate ispinesib, moderate hERG and CYP inhibition. One triphenylbutanamine analogue and ispinesib possess very good bioavailability (51% and 45%, respectively), with the former showing in vivo antitumor growth activity in nude mice xenograft studies.

The structure of the kinesin-1 motor-tail complex reveals the mechanism of autoinhibition.

When not transporting cargo, kinesin-1 is autoinhibited by binding of a tail region to the motor domains, but the mechanism of inhibition is unclear. We report the crystal structure of a motor domain dimer in complex with its tail domain at 2.2 angstroms and compare it with a structure of the motor domain alone at 2.7 angstroms. These structures indicate that neither an induced conformational change nor steric blocking is the cause of inhibition. Instead, the tail cross-links the motor domains at a second position, in addition to the coiled coil. This "double lockdown," by cross-linking at two positions, prevents the movement of the motor domains that is needed to undock the neck linker and release adenosine diphosphate. This autoinhibition mechanism could extend to some other kinesins.

Structure-activity relationship and multidrug resistance study of new S-trityl-L-cysteine derivatives as inhibitors of Eg5.

The mitotic spindle is a validated target for cancer chemotherapy. Drugs such as taxanes and vinca alkaloids specifically target microtubules and cause the mitotic spindle to collapse. However, toxicity and resistance are problems associated with these drugs. Thus, alternative approaches to inhibiting the mitotic spindle are being pursued. These include targeting Eg5, a human kinesin involved in the formation of the bipolar spindle. We previously identified S-trityl-L-cysteine (STLC) as a potent allosteric inhibitor of Eg5. Here, we report the synthesis of a new series of STLC-like compounds with in vitro inhibition in the low nanomolar range. We also performed a multidrug resistance study in cell lines overexpressing P-glycoprotein and showed that some of these inhibitors may have the potential to overcome susceptibility to this efflux pump. Finally, we performed molecular docking of the compounds and determined the structures of two Eg5-inhibitor complexes to explain the structure-activity relationship of these compounds.

Structure-activity relationships and molecular docking of novel dihydropyrimidine-based mitotic Eg5 inhibitors.

Dihydropyrimidine-based compounds belong to the first discovered inhibitors of the human mitotic kinesin Eg5. Although they are used by many research groups as model compounds for chemical genetics, considerably less emphasis has been placed on the improvement of this type of inhibitor, with the exception of two recent studies. Dihydropyrimidines can be divided into class I (analogues that bind in the S configuration) and class II type inhibitors, which bind in the R configuration. Herein we report the synthesis and optimization of novel class II type dihydropyrimidines using a combination of in vitro and docking techniques.

Structural basis for inhibition of Eg5 by dihydropyrimidines: stereoselectivity of antimitotic inhibitors enastron, dimethylenastron and fluorastrol.

Human kinesin Eg5, which plays an essential role in mitosis by establishing the bipolar spindle, has proven to be an interesting drug target for the development of cancer chemotherapeutics. Here, we report the crystal structures of the Eg5 motor domain complexed with enastron, dimethylenastron, and fluorastrol. By comparing these structures to that of monastrol and mon-97, we identified the main reasons for increased potency of these new inhibitors, namely the better fit of the ligand to the allosteric binding site and the addition of fluorine atoms. We also noticed preferential binding of the S-enantiomer of enastron and dimethylenastron to Eg5, while the R-enantiomer of fluorastrol binds preferentially to Eg5. In addition, we performed a multidrug resistance (MDR) study in cell lines overexpressing P-glycoprotein (Pgp). We showed that one of these inhibitors may have the potential to overcome susceptibility to this efflux pump and hence overcome common resistance associated with tubulin-targeting drugs.

An allosteric transition trapped in an intermediate state of a new kinesin-inhibitor complex.

Human kinesin Eg5 plays an essential role in mitosis by separating duplicated centrosomes and establishing the bipolar spindle. Eg5 is an interesting drug target for the development of cancer chemotherapy, with seven inhibitors already in clinical trials. In the present paper, we report the crystal structure of the Eg5 motor domain complexed with a potent antimitotic inhibitor STLC (S-trityl-L-cysteine) to 2.0 A (1 A=0.1 nm) resolution. The Eg5-STLC complex crystallizes in space group P3(2) with three molecules per asymmetric unit. Two of the molecules reveal the final inhibitor-bound state of Eg5, whereby loop L5 has swung downwards to close the inhibitor-binding pocket, helix alpha4 has rotated by approx. 15 degrees and the neck-linker has adopted a docked conformation. The third molecule, however, revealed an unprecedented intermediate state, whereby local changes at the inhibitor-binding pocket have not propagated to structural changes at the switch II cluster and neck-linker. This provides structural evidence for the sequence of drug-induced conformational changes.