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 IC value of 10.8 nM in a biochemical assay.

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.

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.

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.

Prostate Cancer Metastases Are Strongly Inhibited by Agonistic Epha2 Ligands in an Orthotopic Mouse Model.

The EphA2 tyrosine kinase receptor is highly expressed in several types of solid tumors. In our recent studies, we targeted EphA2 in pancreatic cancer with agonistic agents and demonstrated that suppression of EphA2 significantly reduced cancer-cell migration in cell-based assays. In the present study, we focused on targeting EphA2 in prostate cancer.

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.

Potential Therapeutic Targeting of Coronavirus Spike Glycoprotein Priming.

Processing of certain viral proteins and bacterial toxins by host serine proteases is a frequent and critical step in virulence. The coronavirus spike glycoprotein contains three (S1, S2, and S2') cleavage sites that are processed by human host proteases. The exact nature of these cleavage sites, and their respective processing proteases, can determine whether the virus can cross species and the level of pathogenicity.

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.

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.

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).

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.

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.

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.

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.

Human resistin protects against endotoxic shock by blocking LPS-TLR4 interaction.

Helminths trigger multiple immunomodulatory pathways that can protect from sepsis. Human resistin (hRetn) is an immune cell-derived protein that is highly elevated in helminth infection and sepsis. However, the function of hRetn in sepsis, or whether hRetn influences helminth protection against sepsis, is unknown.

hBfl-1/hNOXA Interaction Studies Provide New Insights on the Role of Bfl-1 in Cancer Cell Resistance and for the Design of Novel Anticancer Agents.

Upregulation of antiapoptotic Bcl-2 proteins in certain tumors confers cancer cell resistance to chemotherapy or radiations. Members of the antiapoptotic Bcl-2 proteins, including Bcl-2, Mcl-1, Bcl-xL, Bcl-w, and Bfl-1, inhibit apoptosis by selectively binding to conserved α-helical regions, named BH3 domains, of pro-apoptotic proteins such as Bim, tBid, Bad, or NOXA. Five antiapoptotic proteins have been identified that interact with various selectivity with BH3 containing pro-apoptotic counterparts.

Theranostic Nanocages for Imaging and Photothermal Therapy of Prostate Cancer Cells by Active Targeting of Neuropeptide-Y Receptor.

Gold nanocages (AuNCs) have been shown to be a useful tool for harnessing imaging and hyperthermia therapy of cancer, thanks to their unique optical properties, low toxicity, and facile surface functionalization. Herein, we use AuNCs for selective targeting of prostate cancer cells (PC3) via specific interaction between neuropeptide Y (NPY) receptor and three different NPY analogs conjugated to AuNCs. Localized surface plasmon resonance band of the nanoconjugates was set around 800 nm, which is appropriate for in vivo applications.

"Blind" targeting in action: From phage display to breast cancer cell targeting with peptide-gold nanoconjugates.

Tumor homing peptides (THPs) specific for a representative breast cancer cell line (MCF-7) were carefully selected basing on a phage-displayed peptide library freely available on the web, namely the "TumorHoPe: A Database of Tumor Homing Peptides". The selected THPs were synthesized and evaluated in terms of their affinity toward MCF-7 cells. Out of 5 tested THPs, 3 best-performing peptide sequences and 1 scrambled sequence were separately conjugated to spherical gold nanoparticles yielding stable nanoconjugates.

Picomolar Inhibition of Plasmepsin V, an Essential Malaria Protease, Achieved Exploiting the Prime Region.

Malaria is an infectious disease caused by Plasmodium parasites. It results in an annual death-toll of ~ 600,000. Resistance to all medications currently in use exists, and novel antimalarial drugs are urgently needed.

NMR fingerprints of the drug-like natural-product space identify iotrochotazine A: a chemical probe to study Parkinson's disease.

The NMR spectrum of a mixture of small molecules is a fingerprint of all of its components. Herein, we present an NMR fingerprint method that takes advantage of the fact that fractions contain simplified NMR profiles, with minimal signal overlap, to allow the identification of unique spectral patterns. The approach is exemplified in the identification of a novel natural product, iotrochotazine A (1), sourced from an Australian marine sponge Iotrochota sp.