USP7 small-molecule inhibitors interfere with ubiquitin binding.

The ubiquitin system regulates essential cellular processes in eukaryotes. Ubiquitin is ligated to substrate proteins as monomers or chains and the topology of ubiquitin modifications regulates substrate interactions with specific proteins. Thus ubiquitination directs a variety of substrate fates including proteasomal degradation. Deubiquitinase enzymes cleave ubiquitin from substrates and are implicated in disease; for example, ubiquitin-specific protease-7 (USP7) regulates stability of the p53 tumour suppressor and other proteins critical for tumour cell survival. However, developing selective deubiquitinase inhibitors has been challenging and no co-crystal structures have been solved with small-molecule inhibitors. Here, using nuclear magnetic resonance-based screening and structure-based design, we describe the development of selective USP7 inhibitors GNE-6640 and GNE-6776. These compounds induce tumour cell death and enhance cytotoxicity with chemotherapeutic agents and targeted compounds, including PIM kinase inhibitors. Structural studies reveal that GNE-6640 and GNE-6776 non-covalently target USP7 12 Å distant from the catalytic cysteine. The compounds attenuate ubiquitin binding and thus inhibit USP7 deubiquitinase activity. GNE-6640 and GNE-6776 interact with acidic residues that mediate hydrogen-bond interactions with the ubiquitin Lys48 side chain, suggesting that USP7 preferentially interacts with and cleaves ubiquitin moieties that have free Lys48 side chains. We investigated this idea by engineering di-ubiquitin chains containing differential proximal and distal isotopic labels and measuring USP7 binding by nuclear magnetic resonance. This preferential binding protracted the depolymerization kinetics of Lys48-linked ubiquitin chains relative to Lys63-linked chains. In summary, engineering compounds that inhibit USP7 activity by attenuating ubiquitin binding suggests opportunities for developing other deubiquitinase inhibitors and may be a strategy more broadly applicable to inhibiting proteins that require ubiquitin binding for full functional activity.

Attenuation of the Reaction of Michael Acceptors with Biologically Important Nucleophiles.

ß-Elimination of drugs tethered to macromolecular carbamates provides a platform for drug half-life extension. However, the macromolecular Michael acceptor products formed upon drug release can potentially react with biological amines and thiols and may raise concerns about safety. We desired to mitigate this possibility by developing linkers that have predictable rates of ß-elimination but suppressed rates of nucleophilic addition to their Michael acceptor products. We prepared Michael acceptor products of ß-eliminative linkers that contained a methyl group at the Cß carbon or a gem-dimethyl group at the Cγ carbon and studied the kinetics of their reactions with the most prevalent biological nucleophiles-amine and thiol groups. Aza-Michael reactions with glycine are slowed about 20-fold by methylation of the ß-carbon and 175-fold with a gem-dimethyl group at the γ-carbon. Likewise, addition of the glutathione thiol to γ-gem-dimethyl Michael acceptors was retarded 7-24-fold compared to parent unsubstituted linkers. It was estimated that in an in vivo environment of ∼0.5 mM macromolecular thiols or ∼20 mM macromolecular amines-as in plasma-the reaction half-life of a typical Michael acceptor with a γ-gem-dimethyl linker could exceed 3 years for thiols or 25 years for amines. We also prepared a large series of γ-gem-dimethyl ß-eliminative linkers and showed excellent structure-activity relationships of elimination rates with corresponding unsubstituted parent linkers. Finally, we compared the first-generation unsubstituted and new gem-dimethyl ß-eliminative linkers in a once-monthly drug delivery system of a 39 amino acid peptide. Both linkers provided the desired half-life extension of the peptide, but the Michael acceptor formed from the gem-dimethyl linker was much less reactive. We conclude that the γ-gem-dimethyl ß-eliminative linkers provide high flexibility and greatly reduce potential reactions of Michael acceptor products with biologically important nucleophiles.

Approach for Half-Life Extension of Small Antibody Fragments That Does Not Affect Tissue Uptake.

The utility of antigen-binding antibody fragments is often limited by their short half-lives. Half-life extension of such fragments is usually accomplished by attachment or binding to high-molecular-weight carriers that reduce the renal elimination rate. However, the higher hydrodynamic radius results in greater confinement in the vascular compartment and, thus, lower tissue distribution. We have developed a chemically controlled drug delivery system in which the drug is covalently attached to hydrogel microspheres by a self-cleaving ß-eliminative linker; upon subcutaneous injection, the t1/2,ß of the released drug acquires the t1/2 of linker cleavage. In the present work, we compared the pharmacokinetics of an anti-TNFα scFv, the same scFv attached to 40 kDa PEG by a stable linker, and the scFv attached to hydrogel microspheres by a self-cleaving linker. We also developed a general approach for the selective attachment of ß-eliminative linkers to the N-termini of proteins. In rats, the scFv had a t1/2,ß of 4 h and a high volume of distribution at steady state (Vd,SS), suggesting extensive tissue distribution. The PEG-scFv conjugate had an increased t1/2,ß of about 2 days but showed a reduced Vd,SS that was similar to the plasma volume. In contrast, the tissue-penetrable scFv released from the hydrogel system had a t1/2,ß of about 2 weeks. Thus, the cleavable microsphere-scFv conjugate releases its protein cargo with a prolonged half-life comparable to that of most full-length mAbs and in a form that has the high tissue distribution characteristic of smaller mAb fragments. Other antigen-binding antibody fragments should be amenable to the half-life extension approach described here.

Biodegradable tetra-PEG hydrogels as carriers for a releasable drug delivery system.

We have developed an approach to prepare drug-releasing Tetra-PEG hydrogels with exactly four cross-links per monomer. The gels contain two cleavable ß-eliminative linkers: one for drug attachment that releases the drug at a predictable rate, and one with a longer half-life placed in each cross-link to control biodegradation. Thus, the system can be optimized to release the drug before significant gel degradation occurs. The synthetic approach involves placing a heterobifunctional connector at each end of a four-arm PEG prepolymer; four unique end-groups of the resultant eight-arm prepolymer are used to tether a linker-drug, and the other four are used for polymerization with a second four-arm PEG. Three different orthogonal reactions that form stable triazoles, diazines, or oximes have been used for tethering the drug to the PEG and for cross-linking the polymer. Three formats for preparing hydrogel-drug conjugates are described that either polymerize preformed PEG-drug conjugates or attach the drug postpolymerization. Degradation of drug-containing hydrogels proceeds as expected for homogeneous Tetra-PEG gels with minimal degradation occurring in early phases and sharp, predictable reverse gelation times. The minimal early degradation allows design of gels that show almost complete drug release before significant gel-drug fragments are released.

Broad-spectrum allosteric inhibition of herpesvirus proteases.

Herpesviruses rely on a homodimeric protease for viral capsid maturation. A small molecule, DD2, previously shown to disrupt dimerization of Kaposi's sarcoma-associated herpesvirus protease (KSHV Pr) by trapping an inactive monomeric conformation and two analogues generated through carboxylate bioisosteric replacement (compounds 2 and 3) were shown to inhibit the associated proteases of all three human herpesvirus (HHV) subfamilies (α, ß, and γ). Inhibition data reveal that compound 2 has potency comparable to or better than that of DD2 against the tested proteases. Nuclear magnetic resonance spectroscopy and a new application of the kinetic analysis developed by Zhang and Poorman [Zhang, Z. Y., Poorman, R. A., et al. (1991) J. Biol. Chem. 266, 15591-15594] show DD2, compound 2, and compound 3 inhibit HHV proteases by dimer disruption. All three compounds bind the dimer interface of other HHV proteases in a manner analogous to binding of DD2 to KSHV protease. The determination and analysis of cocrystal structures of both analogues with the KSHV Pr monomer verify and elaborate on the mode of binding for this chemical scaffold, explaining a newly observed critical structure-activity relationship. These results reveal a prototypical chemical scaffold for broad-spectrum allosteric inhibition of human herpesvirus proteases and an approach for the identification of small molecules that allosterically regulate protein activity by targeting protein-protein interactions.

Cyanopyrrolidine Inhibitors of Ubiquitin Specific Protease 7 Mediate Desulfhydration of the Active-Site Cysteine.

Ubiquitin specific protease 7 (USP7) regulates the protein stability of key cellular regulators in pathways ranging from apoptosis to neuronal development, making it a promising therapeutic target. Here we used an engineered, activated variant of the USP7 catalytic domain to perform structure-activity studies of electrophilic peptidomimetic inhibitors. Employing this USP7 variant, we found that inhibitors with a cyanopyrrolidine warhead unexpectedly promoted a ß-elimination reaction of the initial covalent adducts, thereby converting the active-site cysteine residue to dehydroalanine. We determined that this phenomenon is specific for the USP7 catalytic cysteine and that structural features of the inhibitor and protein microenvironment impact elimination rates. Using comprehensive docking studies, we propose that the characteristic conformational dynamics of USP7 allow access to conformations that promote the ligand-induced elimination. Unlike in conventional reversible-covalent inhibition, the compounds described here irreversibly destroy a catalytic residue while simultaneously converting the inhibitor to a nonelectrophilic byproduct. Accordingly, this unexpected finding expands the scope of covalent inhibitor modalities and offers intriguing insights into enzyme-inhibitor dynamics.

PET Imaging of the EPR Effect in Tumor Xenografts Using Small 15 nm Diameter Polyethylene Glycols Labeled with Zirconium-89.

The goal was to develop and characterize a companion diagnostic for the releasable PEG40kDa∼SN-38 oncology drug, PLX038, that would identify tumors susceptible to high accumulation of PLX038. PEG conjugates of the zirconium ligand desferroxamine B (DFB) of similar size and charge to PLX038 were prepared that contained one or four DFB, as well as one that contained three SN-38 moieties and one DFB. Uptake and associated kinetic parameters of the 89Zr-labeled nanocarriers were determined in tumor and normal tissues in mice using µPET/CT imaging. The data were fit to physiologically based pharmacokinetic models to simulate the mass-time profiles of distribution of conjugates in the tissues of interest. The time-activity curves for normal tissues showed high levels at the earliest time of measurement due to vascularization, followed by a monophasic loss. In tumors, levels were initially lower than in normal tissues but increased to 9% to 14% of injected dose over several days. The efflux half-life in tumors was very long, approximately 400 hours, and tumor levels remained at about 10% injected dose 9 days after injection. Compared with diagnostic liposomes, the PEG nanocarriers have a longer serum half-life, are retained in tumors at higher levels, remain there longer, and afford higher tumor exposure. The small PEG40kDa nanocarriers studied here show properties for passive targeting of tumors that are superior than most nanoparticles and might be effective probes to identify tumors susceptible to similar size therapeutic nanocarriers such as PLX038.

Selectively Modulating Conformational States of USP7 Catalytic Domain for Activation.

Ubiquitin-specific protease 7 (USP7) deubiquitinase activity is controlled by a number of regulatory factors, including stimulation by intramolecular accessory domains. Alone, the USP7 catalytic domain (USP7cd) shows limited activity and apo USP7cd crystal structures reveal a disrupted catalytic triad. By contrast, ubiquitin-conjugated USP7cd structures demonstrate the canonical cysteine protease active-site geometry; however, the structural features of the USP7cd that stabilize the inactive conformation and the mechanism of transition between inactive and active states remain unclear. Here we use comparative structural analyses, molecular dynamics simulations, and in silico sequence re-engineering via directed sampling by RosettaDesign to identify key molecular determinants of USP7cd activation and successfully engineer USP7cd for improved activity. Full kinetic analysis and multiple X-ray crystal structures of our designs indicate that electrostatic interactions in the distal "switching loop" region and local packing in the hydrophobic core mediate subtle but significant conformational changes that modulate USP7cd activation.

Primary deuterium kinetic isotope effects prolong drug release and polymer biodegradation in a drug delivery system.

We have developed a chemically-controlled drug delivery system in which a drug is covalently attached via a carbamate to hydrogel microspheres using a ß-eliminative linker; rate-determining proton removal from a CH bond adjacent to an electron withdrawing group results in a ß-elimination to cleave the carbamate and release the drug. After subcutaneous injection of the hydrogel-drug conjugate, the drug is slowly released into the systemic circulation and acquires an elimination t1/2,ß that matches the t1/2 of linker cleavage. A similar ß-eliminative linker with a slower cleavage rate is installed into crosslinks of the polymer to trigger gel degradation after drug release. We have now prepared ß-eliminative linkers that contain deuterium in place of the hydrogen whose removal initiates cleavage. In vitro model systems of drug release and degelation show large primary deuterium kinetic isotope effects of kH/kD ~ 2.5 to 3.5. Using a deuterated linker to attach the peptide octreotide to hydrogel-microspheres, the in vivo t1/2,ß of the drug was increased from ~1.5 to 4.5 weeks in the rat. Similarly, the in vivo time to biodegradation of hydrogels with deuterium-containing crosslinks could be extended ~2.5-fold compared to hydrogen-containing counterparts. Thus, the use of primary deuterium kinetic isotope effects in a single platform technology can control rates of ß-elimination reactions in drug release and polymer biodegradation rates.

Surgical sealants with tunable swelling, burst pressures, and biodegradation rates.

We developed two types of polyethylene glycol (PEG)-based surgical sealants, which we have termed the PER and PRO series. In one, the PRO series, an 8-arm PEG containing activated carbonyl end-groups was reacted with a 4-armed amino-PEG. In the second, the PER series, a 4-arm PEG containing bi-functional end groups with four azides and four activated esters was reacted by strain-promoted alkyne-azide cycloaddition with a 4-arm cyclooctyne-PEG to give a near-ideal Tetra-PEG hydrogel. The sealants showed predictably tunable strength, swelling, adhesion, and gelation properties. The gels were compared to commercially available PEG-based sealants and exhibit physical properties equivalent to or better than the standards. Variants of each gel-format were prepared that contained a ß-eliminative cleavable linker in the crosslinks to control degradation rate. Linkers of this type self-cleave with half-lives spanning from hours to years, and offer the unique ability to precisely tune the degradation to match the healing process. In addition, these linkers could serve as cleavable tethers for controlled drug release. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1602-1611, 2017.

A Hydrogel-Microsphere Drug Delivery System That Supports Once-Monthly Administration of a GLP-1 Receptor Agonist.

We have developed a chemically controlled very long-acting delivery system to support once-monthly administration of a peptidic GLP-1R agonist. Initially, the prototypical GLP-1R agonist exenatide was covalently attached to hydrogel microspheres by a self-cleaving ß-eliminative linker; after subcutaneous injection in rats, the peptide was slowly released into the systemic circulation. However, the short serum exenatide half-life suggested its degradation in the subcutaneous depot. We found that exenatide undergoes deamidation at Asn28 with an in vitro and in vivo half-life of approximately 2 weeks. The [Gln28]exenatide variant and exenatide showed indistinguishable GLP-1R agonist activities as well as pharmacokinetic and pharmacodynamic effects in rodents; however, unlike exenatide, [Gln28]exenatide is stable for long periods. Two different hydrogel-[Gln28]exenatide conjugates were prepared using ß-eliminative linkers with different cleavage rates. After subcutaneous injection in rodents, the serum half-lives for the released [Gln28]exenatide from the two conjugates were about 2 weeks and one month. Two monthly injections of the latter in the Zucker diabetic fatty rat showed pharmacodynamic effects indistinguishable from two months of continuously infused exenatide. Pharmacokinetic simulations indicate that the delivery system should serve well as a once-monthly GLP-1R agonist for treatment of type 2 diabetes in humans.

C-15 thiazol-4-yl analogues of (E)-9,10-didehydroepothilone D: synthesis and cytotoxicity.

The syntheses and biological evaluation of six epothilone D analogues are reported. These side-chain variants of the (E)-9,10-didehydroepothilone scaffold contain C-15 thiazole appendages that are derived from bromomethyl ketone intermediates. Although each of these analogues is less cytotoxic than the parent (E)-9,10-didehydroepothilone D, three maintain IC(50) values in the double-digit nanomolar range against both susceptible and resistant cell lines.

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.

Ceapins are a new class of unfolded protein response inhibitors, selectively targeting the ATF6α branch.

The membrane-bound transcription factor ATF6α plays a cytoprotective role in the unfolded protein response (UPR), required for cells to survive ER stress. Activation of ATF6α promotes cell survival in cancer models. We used cell-based screens to discover and develop Ceapins, a class of pyrazole amides, that block ATF6α signaling in response to ER stress. Ceapins sensitize cells to ER stress without impacting viability of unstressed cells. Ceapins are highly specific inhibitors of ATF6α signaling, not affecting signaling through the other branches of the UPR, or proteolytic processing of its close homolog ATF6ß or SREBP (a cholesterol-regulated transcription factor), both activated by the same proteases. Ceapins are first-in-class inhibitors that can be used to explore both the mechanism of activation of ATF6α and its role in pathological settings. The discovery of Ceapins now enables pharmacological modulation all three UPR branches either singly or in combination.

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.

A divergent approach to the myriaporones and tedanolide: completion of the carbon skeleton of myriaporone 1.

[reaction: see text] A linear but concise synthetic approach toward the structurally related natural products myriaporone and tedanolide is reported. The route is highlighted by a stereoselective homoallenylboration and a regio- and chemoselective nitrile oxide cycloaddition. Installation of the (Z)-olefin completed the carbon skeleton of myriaporone 1.

Tailoring small molecules for an allosteric site on procaspase-6.

Although they represent attractive therapeutic targets, caspases have so far proven recalcitrant to the development of drugs targeting the active site. Allosteric modulation of caspase activity is an alternate strategy that potentially avoids the need for anionic and electrophilic functionality present in most active-site inhibitors. Caspase-6 has been implicated in neurodegenerative disease, including Huntington's and Alzheimer's diseases. Herein we describe a fragment-based lead discovery effort focused on caspase-6 in its active and zymogen forms. Fragments were identified for procaspase-6 using surface plasmon resonance methods and subsequently shown by X-ray crystallography to bind a putative allosteric site at the dimer interface. A fragment-merging strategy was employed to produce nanomolar-affinity ligands that contact residues in the L2 loop at the dimer interface, significantly stabilizing procaspase-6. Because rearrangement of the L2 loop is required for caspase-6 activation, our results suggest a strategy for the allosteric control of caspase activation with drug-like small molecules.

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.

Mechanistic and structural understanding of uncompetitive inhibitors of caspase-6.

Inhibition of caspase-6 is a potential therapeutic strategy for some neurodegenerative diseases, but it has been difficult to develop selective inhibitors against caspases. We report the discovery and characterization of a potent inhibitor of caspase-6 that acts by an uncompetitive binding mode that is an unprecedented mechanism of inhibition against this target class. Biochemical assays demonstrate that, while exquisitely selective for caspase-6 over caspase-3 and -7, the compound's inhibitory activity is also dependent on the amino acid sequence and P1' character of the peptide substrate. The crystal structure of the ternary complex of caspase-6, substrate-mimetic and an 11 nM inhibitor reveals the molecular basis of inhibition. The general strategy to develop uncompetitive inhibitors together with the unique mechanism described herein provides a rationale for engineering caspase selectivity.