Structure-Activity Studies of Bis-O-Arylglycolamides: Inhibitors of the Integrated Stress Response.

The integrated stress response comprises multiple signaling pathways for detecting and responding to cellular stress that converge at a single event-the phosphorylation of Ser51 on the α-subunit of eukaryotic translation initiation factor 2 (eIF2α). Phosphorylation of eIF2α (eIF2α-P) results in attenuation of global protein synthesis via the inhibitory effects of eIF2α-P on eIF2B, the guanine exchange factor (GEF) for eIF2. Herein we describe structure-activity relationship (SAR) studies of bis-O-arylglycolamides, first-in-class integrated stress response inhibitors (ISRIB). ISRIB analogues make cells insensitive to the effects of eIF2α-P by activating the GEF activity of eIF2B and allowing global protein synthesis to proceed with residual unphosphorylated eIF2α. The SAR studies described herein support the proposed pharmacology of ISRIB analogues as binding across a symmetrical protein-protein interface formed between protein subunits of the dimeric eIF2B heteropentamer.

Pharmacological dimerization and activation of the exchange factor eIF2B antagonizes the integrated stress response.

The general translation initiation factor eIF2 is a major translational control point. Multiple signaling pathways in the integrated stress response phosphorylate eIF2 serine-51, inhibiting nucleotide exchange by eIF2B. ISRIB, a potent drug-like small molecule, renders cells insensitive to eIF2α phosphorylation and enhances cognitive function in rodents by blocking long-term depression. ISRIB was identified in a phenotypic cell-based screen, and its mechanism of action remained unknown. We now report that ISRIB is an activator of eIF2B. Our reporter-based shRNA screen revealed an eIF2B requirement for ISRIB activity. Our results define ISRIB as a symmetric molecule, show ISRIB-mediated stabilization of activated eIF2B dimers, and suggest that eIF2B4 (δ-subunit) contributes to the ISRIB binding site. We also developed new ISRIB analogs, improving its EC50 to 600 pM in cell culture. By modulating eIF2B function, ISRIB promises to be an invaluable tool in proof-of-principle studies aiming to ameliorate cognitive defects resulting from neurodegenerative diseases.

Protease inhibitors targeting coronavirus and filovirus entry.

In order to gain entry into cells, diverse viruses, including Ebola virus, SARS-coronavirus and the emerging MERS-coronavirus, depend on activation of their envelope glycoproteins by host cell proteases. The respective enzymes are thus excellent targets for antiviral intervention. In cell culture, activation of Ebola virus, as well as SARS- and MERS-coronavirus can be accomplished by the endosomal cysteine proteases, cathepsin L (CTSL) and cathepsin B (CTSB). In addition, SARS- and MERS-coronavirus can use serine proteases localized at the cell surface, for their activation. However, it is currently unclear which protease(s) facilitate viral spread in the infected host. We report here that the cysteine protease inhibitor K11777, ((2S)-N-[(1E,3S)-1-(benzenesulfonyl)-5-phenylpent-1-en-3-yl]-2-{[(E)-4-methylpiperazine-1-carbonyl]amino}-3-phenylpropanamide) and closely-related vinylsulfones act as broad-spectrum antivirals by targeting cathepsin-mediated cell entry. K11777 is already in advanced stages of development for a number of parasitic diseases, such as Chagas disease, and has proven to be safe and effective in a range of animal models. K11777 inhibition of SARS-CoV and Ebola virus entry was observed in the sub-nanomolar range. In order to assess whether cysteine or serine proteases promote viral spread in the host, we compared the antiviral activity of an optimized K11777-derivative with that of camostat, an inhibitor of TMPRSS2 and related serine proteases. Employing a pathogenic animal model of SARS-CoV infection, we demonstrated that viral spread and pathogenesis of SARS-CoV is driven by serine rather than cysteine proteases and can be effectively prevented by camostat. Camostat has been clinically used to treat chronic pancreatitis, and thus represents an exciting potential therapeutic for respiratory coronavirus infections. Our results indicate that camostat, or similar serine protease inhibitors, might be an effective option for treatment of SARS and potentially MERS, while vinyl sulfone-based inhibitors are excellent lead candidates for Ebola virus therapeutics.

Pharmacological brake-release of mRNA translation enhances cognitive memory.

Phosphorylation of the α-subunit of initiation factor 2 (eIF2) controls protein synthesis by a conserved mechanism. In metazoa, distinct stress conditions activate different eIF2α kinases (PERK, PKR, GCN2, and HRI) that converge on phosphorylating a unique serine in eIF2α. This collection of signaling pathways is termed the 'integrated stress response' (ISR). eIF2α phosphorylation diminishes protein synthesis, while allowing preferential translation of some mRNAs. Starting with a cell-based screen for inhibitors of PERK signaling, we identified a small molecule, named ISRIB, that potently (IC50 = 5 nM) reverses the effects of eIF2α phosphorylation. ISRIB reduces the viability of cells subjected to PERK-activation by chronic endoplasmic reticulum stress. eIF2α phosphorylation is implicated in memory consolidation. Remarkably, ISRIB-treated mice display significant enhancement in spatial and fear-associated learning. Thus, memory consolidation is inherently limited by the ISR, and ISRIB releases this brake. As such, ISRIB promises to contribute to our understanding and treatment of cognitive disorders. DOI:http://dx.doi.org/10.7554/eLife.00498.001.

Predicting and improving the membrane permeability of peptidic small molecules.

We evaluate experimentally and computationally the membrane permeability of matched sets of peptidic small molecules bearing natural or bioisosteric unnatural amino acids. We find that the intentional introduction of hydrogen bond acceptor-donor pairs in such molecules can improve membrane permeability while retaining or improving other favorable drug-like properties. We employ an all-atom force field based method to calculate changes in free energy associated with the transfer of the peptidic molecules from water to membrane. This computational method correctly predicts rank order experimental permeability trends within congeneric series and is much more predictive than calculations (e.g., clogP) that do not consider three-dimensional conformation.

Studies towards the synthesis of methionine aminopeptidase inhibitors: diversification utilizing a ROMP-derived coupling reagent.

Efforts to synthesize potential methionine aminopeptidase inhibitors is described. Preliminary SAR and docking studies served as a guide to design the compound libraries. "Chromatography-free" synthesis of various heterocyclic amides was realized by using a high-load, soluble coupling reagent derived via ring-opening metathesis polymerization (ROMP). Subsequent microwave-assisted Suzuki reactions with ortho-substituted arylboronic acids, followed by chromatographic purification afforded a 55-member library in high yields and purities. While the biological testing was not satisfactory, concurrent X-ray crystallography studies revealed key structural features essential for inhibition of methionine aminopeptidase, which directed fruitful results reported in the accompanying manuscript. In addition, in silico Lipinksi profiles and ADME properties of the library are also reported.

Ionic immobilization, diversification, and release: application to the generation of a library of methionine aminopeptidase inhibitors.

Development of an ionic immobilization, diversification, and release method for the generation of methionine aminopeptidase inhibitors is reported. This method involves the immobilization of 5-bromofuran-2-carboxylic acid and 5-bromothiophene-2-carboxylic acid onto PS-BEMP, followed by Suzuki reaction on a resin-bound intermediate and subsequent release to provide products in moderate yields and excellent purities. Compound potencies were evaluated on the Co(II), Mn(II), Ni(II), and Fe(II) forms of Escherichia coli MetAP1. The furoic-acid analogs were found to be Mn(II) selective with IC 50 values in the low micromolar range. Qualitative SAR analysis, supplemented by molecular modeling studies, provides valuable information on structural elements responsible for potency and selectivity.

High-load, soluble oligomeric carbodiimide: synthesis and application in coupling reactions.

A facile preparation of a high-load, soluble oligomeric alkyl cyclohexylcarbodiimide (OACC) reagent via ROM polymerization from commercially available starting materials is described. This reagent is exploited as a coupling reagent for esterification, amidation, and dehydration of carboxylic acids (aliphatic and aromatic) with an assortment of alcohols (aliphatic primary, secondary, and benzylic), thiols, phenols, and amines (aliphatic primary, secondary, benzylic, and aromatic/anilines), respectively. Following the coupling event, precipitation with an appropriate solvent (Et(2)O, MeOH, or EtOAc), followed by filtration through a SPE provides the products in good to excellent yield and purity.

High-load, ROMP-generated oligomeric bis-acid chlorides: design of soluble and insoluble nucleophile scavengers.

[reaction: see text] An efficient strategy for scavenging a host of nucleophiles utilizing an oligomeric bis-acid chloride (OBAC), generated from the ROM polymerization of trans-bicyclo[2.2.1]hept-5-ene-2,3-dicarbonyl dichloride, is described. The reactivity and high load of the OBAC reagent is exploited in the scavenging of amines, alcohols, and thiols that are present in excess following a common benzoylation event. Following the scavenging event, these oligomers can be precipitated with EtOAc and filtered (SiO(2)), leaving benzoylated nucleophiles in excellent yield and purity.