We have recently proposed a novel drug discovery approach based on biophysical screening of focused positional scanning libraries in which each element of the library contained a common binding moiety for the given target or class of targets. In this Letter, we report on the implementation of this approach to target metal containing proteins. In our implementation, we first derived a focused positional scanning combinatorial library of peptide mimetics (of approximately 100,000 compounds) in which each element of the library contained the metal-chelating moiety hydroxamic acid at the C-terminal. Screening of this library by nuclear magnetic resonance spectroscopy in solution allowed the identification of a novel and selective compound series targeting MMP-12. The data supported that our general approach, perhaps applied using other metal chelating agents or other initial binding fragments, may result very effective in deriving novel and selective agents against metalloenzyme.

In this paper, we applied an innovative nuclear magnetic resonance (NMR)-guided screening and ligand design approach, named focused high-throughput screening by NMR (fHTS by NMR), to derive potent, low-molecular-weight ligands capable of mimicking interactions elicited by ephrin ligands on the receptor tyrosine kinase EphA4. The agents bind with nanomolar affinity, trigger receptor activation in cellular assays with motor neurons, and provide remarkable motor neuron protection from amyotrophic lateral sclerosis (ALS) patient-derived astrocytes. Structural studies on the complex between EphA4 ligand-binding domain and a most active agent provide insights into the mechanism of the agents at a molecular level. Together with preliminary in vivo pharmacology studies, the data form a strong foundation for the translation of these agents for the treatment of ALS and potentially other human diseases.

The cell surface receptor CD44 is a glycoprotein belonging to the hyaluronan-binding proteins, termed hyaladherins. CD44 is expressed in a wide variety of isoforms in many cells and, in particular, is present on the surface of malignant cells where it is involved in the onset and progression of cancer. In a first attempt to identify novel CD44-binding agents, we first characterized, with NMR spectroscopic techniques, several agents that were reported to bind to human CD44 (hCD44). To our surprise, however, none of these putative CD44-binding agents, including a peptide that is in phase 2 clinical trials (A6 peptide) and a recently reported fragment hit, were found to interact significantly with recombinant hCD44(21-178). Nonetheless, we further report that a fragment-screening campaign, with solution NMR spectroscopy as the detection method, identified a viable fragment hit that bound in a potentially functional pocket on the surface of CD44, opposite to the hyaluronic acid binding site. We hypothesize that this pocket could be indirectly associated with the cellular and in vivo activity of the A6 peptide, which would provide a novel framework for the possible development of therapeutically viable CD44 antagonists.

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.

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.

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.

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.

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. Cancer cells present various and variable levels of these proteins, making the design of effective apoptosis based therapeutics challenging. Recently, BH3 profiling was introduced as a method to classify cancer cells based on their ability to resist apoptosis following exposure to selected BH3 peptides. However, these studies were based on binding affinities measured with model BH3 peptides and Bcl-2-proteins taken from mouse sequences. While the majority of these interactions are conserved between mice and humans, we found surprisingly that human NOXA binds to human Bfl-1 potently and covalently via conserved Cys residues, with over 2 orders of magnitude increased affinity over hMcl-1. Our data suggest that some assumptions of the original BH3 profiling need to be revisited and that perhaps further targeting efforts should be redirected toward Bfl-1, for which no suitable specific inhibitors or pharmacological tools have been reported. In this regard, we also describe the initial design and characterizations of novel covalent BH3-based agents that potently target Bfl-1. These molecules could provide a novel platform on which to design effective Bfl-1 targeting therapeutics.

Amyotrophic lateral sclerosis (ALS) is a degenerative disease that progressively destroys motor neurons (MNs). Earlier studies identified EphA4, a receptor tyrosine kinase, as a possible disease-modifying gene. The complex interplay between the EphA4 receptor and its ephrin ligands in motor neurons and astrocytes has not yet been fully elucidated and includes a putative pro-apoptotic activity of the unbound receptor compared to ephrin-bound receptor. We recently reported that astrocytes from patients with ALS induce cell death in co-cultured MNs. Here we found that first-generation synthetic EphA4 agonistic agent 123C4, effectively protected MNs when co-cultured with reactive astrocytes from patients with ALS from multiple subgroups (sALS and mutant SOD1). Newer generation and more potent EphA4 agonistic agents 150D4, 150E8, and 150E7 provided effective protection at a lower therapeutic dose. Combined, the data suggest that the development of EphA4 agonistic agents provides potentially a promising therapeutic strategy for patients with ALS.