Histidine-Covalent Stapled Alpha-Helical Peptides Targeting hMcl-1.

Several novel and effective cysteine targeting (Cys) covalent drugs are in clinical use. However, the target area containing a druggable Cys residue is limited. Therefore, methods for creating covalent drugs that target different residues are being looked for; examples of such ligands include those that target the residues lysine (Lys) and tyrosine (Tyr). Though the histidine (His) side chain is more frequently found in protein binding locations and has higher desirable nucleophilicity, surprisingly limited research has been done to specifically target this residue, and there are not many examples of His-targeting ligands that have been rationally designed. In the current work, we created novel stapled peptides that are intended to target hMcl-1 His 252 covalently. We describe the in vitro (biochemical, NMR, and X-ray) and cellular design and characterization of such agents. Our findings further suggest that the use of electrophiles to specifically target His residues is warranted.

Mixture-Based Screening of Focused Combinatorial Libraries by NMR: Application to the Antiapoptotic Protein hMcl-1.

We report on an innovative ligand discovery strategy based on protein NMR-based screening of a combinatorial library of ∼125,000 compounds that was arranged in 96 distinct mixtures. Using sensitive solution protein NMR spectroscopy and chemical perturbation-based screening followed by an iterative synthesis, deconvolutions, and optimization strategy, we demonstrate that the approach could be useful in the identification of initial binding molecules for difficult drug targets, such as those involved in protein-protein interactions. As an application, we will report novel agents targeting the Bcl-2 family protein hMcl-1. The approach is of general applicability and could be deployed as an effective screening strategy for de novo identification of ligands, particularly when tackling targets involved in protein-protein interactions.

Characterization of a Potent and Orally Bioavailable Lys-Covalent Inhibitor of Apoptosis Protein (IAP) Antagonist.

We have recently reported on the use of aryl-fluorosulfates in designing water- and plasma-stable agents that covalently target Lys, Tyr, or His residues in the BIR3 domain of the inhibitor of the apoptosis protein (IAP) family. Here, we report further structural, cellular, and pharmacological characterizations of this agent, including the high-resolution structure of the complex between the Lys-covalent agent and its target, the BIR3 domain of X-linked IAP (XIAP). We also compared the cellular efficacy of the agent in two-dimensional (2D) and three-dimensional (3D) cell cultures, side by side with the clinical candidate reversible IAP inhibitor LCL161. Finally, in vivo pharmacokinetic studies indicated that the agent was long-lived and orally bioavailable. Collectively our data further corroborate that aryl-fluorosulfates, when incorporated correctly in a ligand, can result in Lys-covalent agents with pharmacodynamic and pharmacokinetic properties that warrant their use in the design of pharmacological probes or even therapeutics.

Targefrin: A Potent Agent Targeting the Ligand Binding Domain of EphA2.

Overexpression of the receptor tyrosine kinase EphA2 is invariably associated with poor prognosis and development of aggressive metastatic cancers. Guided by our recently solved X-ray structure of the complex between an agonistic peptide and EphA2-LBD, we report on a novel agent, targefrin, that binds to EphA2-LBD with a 21 nM dissociation constant by isothermal titration calorimetry and presents an IC50 value of 10.8 nM in a biochemical assay. In cell-based assays, a dimeric version of the agent is as effective as the natural dimeric ligands (ephrinA1-Fc) in inducing cellular receptor internalization and degradation in several pancreatic cancer cell lines. When conjugated with chemotherapy, the agents can effectively deliver paclitaxel to pancreatic cancers in a mouse xenograft study. Given the pivotal role of EphA2 in tumor progression, we are confident that the agents reported could be further developed into innovative EphA2-targeting therapeutics.

Lysine Covalent Antagonists of Melanoma Inhibitors of Apoptosis Protein.

We have recently reported on Lys-covalent agents that, based on aryl-sulfonyl fluorides, were designed to target binding site Lys 311 in the X-linked inhibitor of apoptosis protein (XIAP). Similar to XIAP, melanoma-IAP (ML-IAP), a less well-characterized IAP family protein, also presents a lysine residue (Lys 135), which is in a position equivalent to that of Lys 311 of XIAP. On the contrary, two other members of the IAP family, namely, cellular-IAPs (cIAP1 and cIAP2), present a glutamic acid residue in that position. Hence, in the present work, we describe the derivation and characterization of the very first potent ML-IAP Lys-covalent inhibitor with cellular activity. The agent can be used as a pharmacological tool to further validate ML-IAP as a drug target and eventually for the development of ML-IAP-targeted therapeutics.

Effective Tumor Targeting by EphA2-Agonist-Biotin-Streptavidin Conjugates.

We recently reported on a potent synthetic agent, 135H11, that selectively targets the receptor tyrosine kinase, EphA2. While 135H11 possesses a relatively high binding affinity for the ligand-binding domain of EphA2 (Kd~130 nM), receptor activation in the cell required the synthesis of dimeric versions of such agent (namely 135H12). This was expected given that the natural ephrin ligands also need to be dimerized or clustered to elicit agonistic activity in cell. In the present report we investigated whether the agonistic activity of 135H11 could be enhanced by biotin conjugation followed by complex formation with streptavidin. Therefore, we measured the agonistic EphA2 activity of 135H11-biotin (147B5) at various agent/streptavidin ratios, side by side with 135H12, and a scrambled version of 147B5 in pancreatic- and breast-cancer cell lines. The (147B5)n-streptavidin complexes (when n = 2, 3, 4, but not when n = 1) induced a strong receptor degradation effect in both cell lines compared to 135H12 or the (scrambled-147B5)4-streptavidin complex as a control, indicating that multimerization of the targeting agent resulted in an increased ability to cause receptor clustering and internalization. Subsequently, we prepared an Alexa-Fluor-streptavidin conjugate to demonstrate that (147B5)4-AF-streptavidin, but not the scrambled equivalent complex, concentrates in pancreatic and breast cancers in orthotopic nude-mouse models. Hence, we conclude that these novel targeting agents, with proper derivatization with imaging reagents or chemotherapy, can be used as diagnostics, and/or to deliver chemotherapy selectively to EphA2-expressing tumors.

Design, Synthesis, and Structural Characterization of Lysine Covalent BH3 Peptides Targeting Mcl-1.

Modulating disease-relevant protein-protein interactions (PPIs) using pharmacological tools is a critical step toward the design of novel therapeutic strategies. Over the years, however, targeting PPIs has proven a very challenging task owing to the large interfacial areas. Our recent efforts identified possible novel routes for the design of potent and selective inhibitors of PPIs using a structure-based design of covalent inhibitors targeting Lys residues. In this present study, we report on the design, synthesis, and characterizations of the first Lys-covalent BH3 peptide that has a remarkable affinity and selectivity for hMcl-1 over the closely related hBfl-1 protein. Our structural studies, aided by X-ray crystallography, provide atomic-level details of the inhibitor interactions that can be used to further translate these discoveries into novel generation, Lys-covalent pro-apoptotic agents.

Stability and Cell Permeability of Sulfonyl Fluorides in the Design of Lys-Covalent Antagonists of Protein-Protein Interactions.

Recently we reported on aryl-fluorosulfates as possible stable and effective electrophiles for the design of lysine covalent, cell permeable antagonists of protein-protein interactions (PPIs). Here we revisit the use of aryl-sulfonyl fluorides as Lys-targeting moieties, incorporating these electrophiles in XIAP (X-linked inhibitor of apoptosis protein) targeting agents. We evaluated stability in buffer and reactivity with Lys311 of XIAP of various aryl-sulfonyl fluorides using biochemical and biophysical approaches, including displacement assays, mass spectrometry, SDS gel electrophoresis, and denaturation thermal shift measurements. To assess whether these modified electrophilic "warheads" can also react with Tyr, we repeated these evaluations with a Lys311Tyr XIAP mutant. Using a direct cellular assay, we could demonstrate that selected agents are cell permeable and interact covalently with their intended target in cell. These results suggest that certain substituted aryl-sulfonyl fluorides can be useful Lys- or Tyr-targeting electrophiles for the design of covalent pharmacological tools or even future therapeutics targeting protein-protein interactions.

Therapeutic Targeting of Pancreatic Cancer via EphA2 Dimeric Agonistic Agents.

Recently, we reported on potent EphA2 targeting compounds and demonstrated that dimeric versions of such agents can exhibit remarkably increased agonistic activity in cellular assays compared to the monomers. Here we further characterize the activity of dimeric compounds at the structural, biochemical, and cellular level. In particular, we propose a structural model for the mechanism of receptor activation by dimeric agents and characterize the effect of most potent compounds in inducing EphA2 activation and degradation in a pancreatic cancer cell line. These cellular studies indicate that the pro-migratory effects induced by the receptor can be reversed in EphA2 knockout cells, by treatment with either a dimeric natural ligand (ephrinA1-Fc), or by our synthetic agonistic dimers. Based on these data we conclude that the proposed agents hold great potential as possible therapeutics in combination with standard of care, where these could help suppressing a major driver for cell migration and tumor metastases. Finally, we also found that, similar to ephrinA1-Fc, dimeric agents cause a sustained internalization of the EphA2 receptor, hence, with proper derivatizations, these could also be used to deliver chemotherapy selectively to pancreatic tumors.

N-locking stabilization of covalent helical peptides: Application to Bfl-1 antagonists.

Recently, it was reported that tetrapeptides cyclized via lactam bond between the amino terminus and a glutamic residue in position 4 (termed here N-lock) can nucleate helix formation in longer peptides. We applied such strategy to derive N-locked covalent BH3 peptides that were designed to selectively target the anti-apoptotic protein Bfl-1. The resulting agents were soluble in aqueous buffer and displayed a remarkable (low nanomolar) affinity for Bfl-1 and cellular activity. The crystal structure of the complex between such N-locked covalent peptide and Bfl-1 provided insights on the geometry of the N-locking strategy and of the covalent bond between the agent and Bfl-1.

Aryl-fluorosulfate-based Lysine Covalent Pan-Inhibitors of Apoptosis Protein (IAP) Antagonists with Cellular Efficacy.

We have recently investigated the reactivity of aryl-fluorosulfates as warheads to form covalent adducts with Lys, Tyr, and His residues. However, the rate of reaction of aryl-fluorosulfates seemed relatively slow, putting into question their effectiveness to form covalent adducts in cell. Unlike the previously reported agents that targeted a relatively remote Lys residue with respect to the target's binding site, the current agents were designed to more directly juxtapose an aryl-fluorosulfate with a Lys residue that is located within the binding pocket of the BIR3 domain of X-linked inhibitor of apoptosis protein (XIAP). We found that such new agents can effectively and rapidly form a covalent adduct with XIAP-BIR3 in vitro and in cell, approaching the rate of reaction, cellular permeability, and stability that are similar to what attained by acrylamides when targeting Cys residues. Our studies further validate aryl-fluorosulfates as valuable Lys-targeting electrophiles, for the design of inhibitors of both enzymes and protein-protein interactions.

Covalent Inhibitors of Protein-Protein Interactions Targeting Lysine, Tyrosine, or Histidine Residues.

We have recently reported a series of Lys-covalent agents targeting the BIR3 domain of the X-linked inhibitor of apoptosis protein (XIAP) using a benzamide-sulfonyl fluoride warhead. Using XIAP as a model system, we further investigated a variety of additional warheads that can be easily incorporated into binding peptides and analyzed their ability to form covalent adducts with lysine and other amino acids, including tyrosine, histidine, serine, and threonine, using biochemical and biophysical assays. Moreover, we tested aqueous, plasma stability, cell permeability, and cellular efficacy of the most effective agents. These studies identified aryl-fluoro sulfates as likely the most suitable electrophiles to effectively form covalent adducts with Lys, Tyr, and His residues, given that these agents were cell permeable and stable in aqueous buffer and in plasma. Our studies contain a number of general findings that open new possible avenues for the design of potent covalent protein-protein interaction antagonists.

Structure-Based Design of Novel EphA2 Agonistic Agents with Nanomolar Affinity in Vitro and in Cell.

EphA2 overexpression is invariably associated with poor prognosis and development of aggressive metastatic cancers in pancreatic, prostate, lung, ovarian, and breast cancers and melanoma. Recent efforts from our laboratories identified a number of agonistic peptides targeting the ligand-binding domain of the EphA2 receptor. The individual agents, however, were still relatively weak in affinities (micromolar range) that precluded detailed structural studies on the mode of action. Using a systematic optimization of the 12-mer peptide mimetic 123B9, we were able to first derive an agent that displayed a submicromolar affinity for the receptor. This agent enabled cocrystallization with the EphA2 ligand-binding domain providing for the first time the structural basis for their agonistic mechanism of action. In addition, the atomic coordinates of the complex enabled rapid iterations of structure-based optimizations that resulted in a novel agonistic agent, named 135H11, with a nanomolar affinity for the receptor, as demonstrated by in vitro binding assays (isothermal titration calorimetry measurements), and a biochemical displacement assay. As we have recently demonstrated, the cellular activity of these agents is further increased by synthesizing dimeric versions of the compounds. Hence, we report that a dimeric version of 135H11 is extremely effective at low nanomolar concentrations to induce cellular receptor activation, internalization, and inhibition of cell migration in a pancreatic cancer cell line. Given the pivotal role of EphA2 in tumor growth, angiogenesis, drug resistance, and metastasis, these agents, and the associated structural studies, provide significant advancements in the field for the development of novel EphA2-targeting therapeutics or diagnostics.

Design of Potent pan-IAP and Lys-Covalent XIAP Selective Inhibitors Using a Thermodynamics Driven Approach.

Recently we reported that rapid determination of enthalpy of binding can be achieved for a large number of congeneric agents or in combinatorial libraries fairly efficiently. We show that using a thermodynamic Craig plot can be very useful in dissecting the enthalpy and entropy contribution of different substituents on a common scaffold, in order to design potent, selective, or pan-active compounds. In our implementation, the approach identified a critical Lys residue in the BIR3 domain of XIAP. We report for the first time that it is possible to target such residue covalently to attain potent and selective agents. Preliminary cellular studies in various models of leukemia, multiple myeloma, and pancreatic cancers suggest that the derived agents possess a potentially intriguing pattern of activity, especially for cell lines that are resistant to the pan-IAP antagonist and clinical candidate LCL161.

Reduction of Circulating Cancer Cells and Metastases in Breast-Cancer Models by a Potent EphA2-Agonistic Peptide-Drug Conjugate.

EphA2 overexpression has been associated with metastasis in multiple cancer types, including melanomas and ovarian, prostate, lung, and breast cancers. We have recently proposed the development of peptide-drug conjugates (PDCs) using agonistic EphA2-targeting agents, such as the YSA peptide or its optimized version, 123B9. Although our studies indicated that YSA- and 123B9-drug conjugates can selectively deliver cytotoxic drugs to cancer cells in vivo, the relatively low cellular agonistic activities (i.e., the high micromolar concentrations required) of the agents toward the EphA2 receptor remained a limiting factor to the further development of these PDCs in the clinic. Here, we report that a dimeric version of 123B9 can induce receptor activation at nanomolar concentrations. Furthermore, we demonstrated that the conjugation of dimeric 123B9 with paclitaxel is very effective at targeting circulating tumor cells and inhibiting lung metastasis in breast-cancer models. These studies represent an important step toward the development of effective EphA2-targeting PDCs.

Enthalpy-Based Screening of Focused Combinatorial Libraries for the Identification of Potent and Selective Ligands.

In modern drug discovery, the ability of biophysical methods, including nuclear magnetic resonance spectroscopy or surface plasmon resonance, to detect and characterize ligand-protein interactions accurately and unambiguously makes these approaches preferred versus conventional biochemical high-throughput screening of large collections of compounds. Nonetheless, ligand screening strategies that address simultaneously potency and selectivity have not yet been fully developed. In this work, we propose a novel method for screening large collections of combinatorial libraries using enthalpy measurements as a primary screening technique. We demonstrate that selecting binders that are driven by enthalpy (ΔH) results in agents that are not only potent but also more selective for a given target. This general and novel approach, we termed ΔH screening of fPOS (enthalpy screening of focused positional scanning library), combines the principles of focused combinatorial chemistry with rapid calorimetry measurements to efficiently identify potent and selective inhibitors.

Interleaved practice enhances skill learning and the functional connectivity of fronto-parietal networks.

Practice of tasks in an interleaved order generally induces superior learning compared with practicing in a repetitive order, a phenomenon known as the contextual-interference (CI) effect. Increased neural activity during interleaved over repetitive practice has been associated with the beneficial effects of CI. Here, we used psychophysiological interaction (PPI) analysis to investigate whether the neural connectivity of the dorsal premotor (PM) and the dorsolateral prefrontal (DLPFC) cortices changes when motor sequences are acquired through interleaved practice. Sixteen adults practiced a serial reaction time task where a set of three 4-element sequences were arranged in a repetitive or in an interleaved order on 2 successive days. On Day 5, participants were tested with practiced sequences to evaluate retention. A within-subjects design was used so that participants practiced sequences in the other condition (repetitive or interleaved) 2-4 weeks later. Functional magnetic resonance images were acquired during practice and retention. On Day 2 of practice, there was greater inter-regional functional connectivity in the interleaved compared with the repetitive condition for both PM-seeded and DLPFC-seeded connectivity. The increased functional connectivity between both seeded regions and sensorimotor cortical areas correlated with the benefit of interleaved practice during later retention. During retention, a significant PPI effect was found in DLPFC-seeded connectivity, with increased DLPFC-supplementary motor area connectivity correlated with the benefits of interleaved practice. These data suggest that interleaved practice benefits learning by enhancing coordination of sensorimotor cortical regions, and superior performance of sequences learned under CI is characterized by increased functional connectivity in frontal cortex.

Age related differences in the neural substrates of motor sequence learning after interleaved and repetitive practice.

Practice of tasks in an interleaved order generally induces superior retention compared to practicing in a repetitive order. Younger and older adults practiced serial reaction time tasks that were arranged in a repeated or an interleaved order on 2 successive days. Retention was tested on Day 5. For both groups, reaction times in the interleaved condition were slower than the repetitive condition during practice, but the reverse was true during retention on Day 5. After interleaved practice, changes in M1 excitability measured by paired-pulse TMS were greater than after repetitive practice, and this effect was more pronounced in older adults. Moreover, the increased M1 excitability correlated with the benefit of interleaved practice. BOLD signal was also increased for interleaved compared to repetitive practice in both groups. However, the pattern of correlations between increased BOLD during practice and subsequent benefit of the interleaved condition differed by group. In younger adults, dorsolateral-prefrontal activity during practice was related to this benefit, while in older adults, activation in sensorimotor regions and rostral prefrontal cortex during practice correlated with the benefit of interleaving on retention. Older adults may engage compensatory mechanisms during interleaved practice such as increasing sensorimotor recruitment which in turn benefits learning.

Enhanced motor learning in older adults is accompanied by increased bilateral frontal and fronto-parietal connectivity.

We recently demonstrated that older adults can benefit as much as younger adults from learning skills in an interleaved manner. Here we investigate whether optimized learning through interleaved practice (IP) is associated with changes in inter-regional brain connectivity and whether younger and older adults differ in such brain-behavior correlations. Younger and older adults practiced a set of three 4-element motor sequences in a repetitive or in an interleaved order for 2 consecutive days. Retention of the practiced sequences was evaluated 3 days after practice with functional images acquired simultaneously. A within-subject design was used so that subjects practiced sequences in the other condition (repetitive or interleaved) 2-4 weeks later. Using the psychophysiological interaction (PPI) analysis approach, we found that IP led to higher functional connectivity between the right and left dorsal lateral prefrontal cortex (DLPFC) and between the dorsal premotor cortex (PMd) and inferior parietal lobule (IPL) in older adults. Moreover, increased connectivity between these regions was significantly associated with the learning benefits of IP. In contrast, in younger adults, enhanced learning as a result of IP was associated with increased connectivity between DLPFC and the supplementary motor area (SMA) and the inferior frontal gyrus. These data suggest that though younger and older gain similar behavioral benefits from interleaved training, aging may alter the operation of brain networks underlying such optimized learning.

Brain-behavior correlates of optimizing learning through interleaved practice.

Understanding how to make learning more efficient and effective is an important goal in behavioral neuroscience. The notion of "desirable difficulties" asserts that challenges for learners during study result in superior learning. One "desirable difficulty" that has a robust benefit on learning is contextual interference (CI), in which different tasks are practiced in an interleaved order rather than in a repetitive order. This study is the first to combine functional imaging and paired-pulse transcranial magnetic stimulation to analyze the neural basis of the CI effect in skill learning. Difficulty during practice of a serial reaction time task was manipulated by presenting sequences of response locations in a repetitive or an interleaved order. Participants practiced 3 sequences for 2 days and were tested on day 5 to examine sequence-specific learning. During practice, slower response times (RT), greater frontal-parietal blood-oxygen-level-dependent (BOLD) signal, and higher motor cortex (M1) excitability were found in the interleaved condition compared to the repetitive condition. Consistent with the CI effect, we found faster RT, decreased BOLD signal in frontal-parietal regions, and greater M1 excitability during the day 5 retention task when subjects had practiced interleaved sequences. Correlation analyses indicated that greater BOLD signal in contralateral sensorimotor region and M1 excitability during interleaved practice were interrelated. Furthermore, greater BOLD signal in prefrontal, premotor and parietal areas and greater M1 excitability during interleaved practice correlated with the benefit of interleaved practice on retention. This demonstrates that interleaved practice induces interrelated changes in both cortical hemodynamic responses and M1 excitability, which likely index the formation of enhanced memory traces and efficient long-term retrieval.