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

Targeting ML-IAP for the Design of Cancer Therapeutics

Metastatic malignant melanoma is the leading cause of skin cancer-related death with a 5-year survival rate smaller than 19%. Most malignant melanomas overexpress an oncogene called melanoma inhibitor of apoptosis (ML-IAP), a member of inhibitor of apoptosis (IAP) proteins, that also include oncogenic proteins, XIAP, cIAP1, and cIAP2. These proteins render cancer cells resistant to apoptosis induced by anti-cancer therapies. ML-IAP is overexpressed in melanoma and several other solid tumors, but it is not present in normal adult tissues, making it potentially an ideal target for novel apoptosis-based therapies. However, studies to validate its potential as a therapeutic target have been hampered by the lack of potent and selective pharmacological inhibitors. To this end, we first characterized IAP antagonists that have been recently designed to mimic the interactions between an endogenous IAP antagonist, namely the second mitochondria-derived activator of caspases protein (SMAC), and IAPs. SMAC pro-apoptotic activity is based on a conserved IAP-binding motif of sequence Alanine-Valine-Proline-Isoleucine/Phenylalanine (AVPI/F). This tetra-peptide releases caspases that were sequestered by IAPs, thus restoring apoptosis. Several AVPF mimetics have been reported that target all IAPs indiscriminately, making it difficult to dissect the role of one oncogenic IAP versus another using these available pharmacological inhibitors.Hence, first we employed a highly innovative structure-driven approach to target covalently a nucleophilic Lysine (Lys) residue present in the AVPI/F binding site of both ML-IAP and XIAP, but not in cIAP1 or cIAP2. Pioneering the use of certain Lys-targeting electrophiles including aryl-sulfonyl fluorides and aryl-fluorosulfates, and exploiting subtle structural differences in the sub-pockets that accommodate the third and the fourth residue of AVPF between XIAP and ML-IAP, we were able to derive agent 142I5 as a first-in-class potent and selective Lys-covalent ML-IAP inhibitor. We demonstrate that the ML-IAP targeting agent 142I5 is as effective as pan-IAP inhibitors in restoring apoptosis in apoptosis resistant melanoma cell lines. In summary, we derived an innovative and unprecedented pharmacological tool targeting ML-IAP covalently that can be used to further characterize the role of this oncogene in cancer resistance and could provide a valuable steppingstone for the development of novel apoptosis-based therapeutics

Targeting ML-IAP for the Design of Cancer Therapeutics

Metastatic malignant melanoma is the leading cause of skin cancer-related death with a 5-year survival rate smaller than 19%. Most malignant melanomas overexpress an oncogene called melanoma inhibitor of apoptosis (ML-IAP), a member of inhibitor of apoptosis (IAP) proteins, that also include oncogenic proteins, XIAP, cIAP1, and cIAP2. These proteins render cancer cells resistant to apoptosis induced by anti-cancer therapies. ML-IAP is overexpressed in melanoma and several other solid tumors, but it is not present in normal adult tissues, making it potentially an ideal target for novel apoptosis-based therapies. However, studies to validate its potential as a therapeutic target have been hampered by the lack of potent and selective pharmacological inhibitors. To this end, we first characterized IAP antagonists that have been recently designed to mimic the interactions between an endogenous IAP antagonist, namely the second mitochondria-derived activator of caspases protein (SMAC), and IAPs. SMAC pro-apoptotic activity is based on a conserved IAP-binding motif of sequence Alanine-Valine-Proline-Isoleucine/Phenylalanine (AVPI/F). This tetra-peptide releases caspases that were sequestered by IAPs, thus restoring apoptosis. Several AVPF mimetics have been reported that target all IAPs indiscriminately, making it difficult to dissect the role of one oncogenic IAP versus another using these available pharmacological inhibitors.Hence, first we employed a highly innovative structure-driven approach to target covalently a nucleophilic Lysine (Lys) residue present in the AVPI/F binding site of both ML-IAP and XIAP, but not in cIAP1 or cIAP2. Pioneering the use of certain Lys-targeting electrophiles including aryl-sulfonyl fluorides and aryl-fluorosulfates, and exploiting subtle structural differences in the sub-pockets that accommodate the third and the fourth residue of AVPF between XIAP and ML-IAP, we were able to derive agent 142I5 as a first-in-class potent and selective Lys-covalent ML-IAP inhibitor. We demonstrate that the ML-IAP targeting agent 142I5 is as effective as pan-IAP inhibitors in restoring apoptosis in apoptosis resistant melanoma cell lines. In summary, we derived an innovative and unprecedented pharmacological tool targeting ML-IAP covalently that can be used to further characterize the role of this oncogene in cancer resistance and could provide a valuable steppingstone for the development of novel apoptosis-based therapeutics

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

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

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

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 (Δ<i>H</i>) results in agents that are not only potent but also more selective for a given target. This general and novel approach, we termed Δ<i>H</i> screening of <i>f</i>POS (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

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

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

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