Bis-sulfonamido-2-phenylbenzoxazoles Validate the GroES/EL Chaperone System as a Viable Antibiotic Target.

We recently reported on small-molecule inhibitors of the GroES/GroEL chaperone system as potential antibiotics against Escherichia coli and the ESKAPE pathogens but were unable to establish GroES/GroEL as the cellular target, leading to cell death. In this study, using two of our most potent bis-sulfonamido-2-phenylbenzoxazoles (PBZs), we established the binding site of the PBZ molecules using cryo-EM and found that GroEL was the cellular target responsible for the mode of action. Cryo-EM revealed that PBZ1587 binds at the GroEL ring-ring interface (RRI). A cellular reporter assay confirmed that PBZ1587 engaged GroEL in cells, but cellular rescue experiments showed potential off-target effects. This prompted us to explore a closely related analogue, PBZ1038, which is also bound to the RRI. Biochemical characterization showed potent inhibition of Gram-negative chaperonins but much lower potency of chaperonin from a Gram-positive organism, Enterococcus faecium. A cellular reporter assay showed that PBZ1038 also engaged GroEL in cells and that the cytotoxic phenotype could be rescued by a chromosomal copy of E. faecium GroEL/GroES or by expressing a recalcitrant RRI mutant. These data argue that PBZ1038's antimicrobial action is exerted through inhibition of GroES/GroEL, validating this chaperone system as an antibiotic target.

Human Hsp70 Substrate-Binding Domains Recognize Distinct Client Proteins.

The 13 Hsp70 proteins in humans act on unique sets of substrates with diversity often being attributed to J-domain-containing protein (Hsp40 or JDP) cofactors. We were therefore surprised to find drastically different binding affinities for Hsp70-peptide substrates, leading us to probe substrate specificity among the 8 canonical Hsp70s from humans. We used peptide arrays to characterize Hsp70 binding and then mined these data using machine learning to develop an algorithm for isoform-specific prediction of Hsp70 binding sequences. The results of this algorithm revealed recognition patterns not predicted based on local sequence alignments. We then showed that none of the human isoforms can complement heat-shocked DnaK knockout Escherichia coli cells. However, chimeric Hsp70s consisting of the human nucleotide-binding domain and the substrate-binding domain of DnaK complement during heat shock, providing further evidence in vivo of the divergent function of the Hsp70 substrate-binding domains. We also demonstrated that the differences in heat shock complementation among the chimeras are not due to loss of DnaJ binding. Although we do not exclude JDPs as additional specificity factors, our data demonstrate substrate specificity among the Hsp70s, which has important implications for inhibitor development in cancer and neurodegeneration.

Engaging a Non-catalytic Cysteine Residue Drives Potent and Selective Inhibition of Caspase-6.

Caspases are a family of cysteine-dependent proteases with important cellular functions in inflammation and apoptosis, while also implicated in human diseases. Classical chemical tools to study caspase functions lack selectivity for specific caspase family members due to highly conserved active sites and catalytic machinery. To overcome this limitation, we targeted a non-catalytic cysteine residue (C264) unique to caspase-6 (C6), an enigmatic and understudied caspase isoform. Starting from disulfide ligands identified in a cysteine trapping screen, we used a structure-informed covalent ligand design to produce potent, irreversible inhibitors (3a) and chemoproteomic probes (13-t) of C6 that exhibit unprecedented selectivity over other caspase family members and high proteome selectivity. This approach and the new tools described will enable rigorous interrogation of the role of caspase-6 in developmental biology and in inflammatory and neurodegenerative diseases.

Physachenolide C is a Potent, Selective BET Inhibitor.

A pulldown using a biotinylated natural product of interest in the 17ß-hydroxywithanolide (17-BHW) class, physachenolide C (PCC), identified the bromodomain and extra-terminal domain (BET) family of proteins (BRD2, BRD3, and BRD4), readers of acetyl-lysine modifications and regulators of gene transcription, as potential cellular targets. BROMOscan bromodomain profiling and biochemical assays support PCC as a BET inhibitor with increased selectivity for bromodomain (BD)-1 of BRD3 and BRD4, and X-ray crystallography and NMR studies uncovered specific contacts that underlie the potency and selectivity of PCC toward BRD3-BD1 over BRD3-BD2. PCC also displays characteristics of a molecular glue, facilitating proteasome-mediated degradation of BRD3 and BRD4. Finally, PCC is more potent than other withanolide analogues and gold-standard pan-BET inhibitor (+)-JQ1 in cytotoxicity assays across five prostate cancer (PC) cell lines regardless of androgen receptor (AR)-signaling status.

Discovery and Development of a Selective Inhibitor of the ER Resident Chaperone Grp78.

A recent study illustrated that a fluorescence polarization assay can be used to identify substrate-competitive Hsp70 inhibitors that can be isoform-selective. Herein, we use that assay in a moderate-throughput screen and report the discovery of a druglike amino-acid-based inhibitor with reasonable specificity for the endoplasmic reticular Hsp70, Grp78. Using traditional medicinal chemistry approaches, the potency and selectivity were further optimized through structure-activity relationship (SAR) studies in parallel assays for six of the human Hsp70 isoforms. The top compounds were all tested against a panel of cancer cell lines and disappointingly showed little effect. The top-performing compound, 8, was retested using a series of endoplasmic reticulum (ER) stress-inducing agents and found to synergize with these agents. Finally, 8 was tested in a spheroid tumor model and found to be more potent than in two-dimensional models. The optimized Grp78 inhibitors are the first reported isoform-selective small-molecule-competitive inhibitors of an Hsp70-substrate interaction.

Heat shock protein Grp78/BiP/HspA5 binds directly to TDP-43 and mitigates toxicity associated with disease pathology.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with no cure or effective treatment in which TAR DNA Binding Protein of 43 kDa (TDP-43) abnormally accumulates into misfolded protein aggregates in affected neurons. It is widely accepted that protein misfolding and aggregation promotes proteotoxic stress. The molecular chaperones are a primary line of defense against proteotoxic stress, and there has been long-standing interest in understanding the relationship between chaperones and aggregated protein in ALS. Of particular interest are the heat shock protein of 70 kDa (Hsp70) family of chaperones. However, defining which of the 13 human Hsp70 isoforms is critical for ALS has presented many challenges. To gain insight into the specific Hsp70 that modulates TDP-43, we investigated the relationship between TDP-43 and the Hsp70s using proximity-dependent biotin identification (BioID) and discovered several Hsp70 isoforms associated with TDP-43 in the nucleus, raising the possibility of an interaction with native TDP-43. We further found that HspA5 bound specifically to the RNA-binding domain of TDP-43 using recombinantly expressed proteins. Moreover, in a Drosophila strain that mimics ALS upon TDP-43 expression, the mRNA levels of the HspA5 homologue (Hsc70.3) were significantly increased. Similarly we observed upregulation of HspA5 in prefrontal cortex neurons from human ALS patients. Finally, overexpression of HspA5 in Drosophila rescued TDP-43-induced toxicity, suggesting that upregulation of HspA5 may have a compensatory role in ALS pathobiology.

Caspase-6-cleaved tau is relevant in Alzheimer's disease and marginal in four-repeat tauopathies: Diagnostic and therapeutic implications.

AIM: Tau truncation (tr-tau) by active caspase-6 (aCasp-6) generates tau fragments that may be toxic. Yet the relationship between aCasp-6, different forms of tr-tau and hyperphosphorylated tau (p-tau) accumulation in human brains with Alzheimer's disease (AD) and other tauopathies remains unclear. METHODS: We generated two neoepitope monoclonal antibodies against tr-tau sites (D402 and D13) targeted by aCasp-6. Then, we used five-plex immunofluorescence to quantify the neuronal and astroglial burden of aCasp-6, tr-tau, p-tau and their co-occurrence in healthy controls, AD and primary tauopathies. RESULTS: Casp-6 activation was strongest in AD and Pick's disease (PiD) but almost absent in 4-repeat (4R) tauopathies. In neurons, the tr-tau burden was much more abundant in AD and PiD than in 4R tauopathies and disproportionally higher when normalising by p-tau pathology. Tr-tau astrogliopathy was detected in low numbers in 4R tauopathies. Unexpectedly, about half of tr-tau positive neurons in AD and PiD lacked p-tau aggregates, a finding we confirmed using several p-tau antibodies. CONCLUSIONS: Early modulation of aCasp-6 to reduce tr-tau pathology is a promising therapeutic strategy for AD and PiD but is unlikely to benefit 4R tauopathies. The large percentage of tr-tau-positive neurons lacking p-tau suggests that many vulnerable neurons to tau pathology go undetected when using conventional p-tau antibodies. Therapeutic strategies against tr-tau pathology could be necessary to modulate the extent of tau abnormalities in AD. The disproportionally higher burden of tr-tau in AD and PiD supports the development of biofluid biomarkers against tr-tau to detect AD and PiD and differentiate them from 4R tauopathies at a patient level.

Allosteric differences dictate GroEL complementation of E. coli.

GroES/GroEL is the only bacterial chaperone essential under all conditions, making it a potential antibiotic target. Rationally targeting ESKAPE GroES/GroEL as an antibiotic strategy necessitates studying their structure and function. Herein, we outline the structural similarities between Escherichia coli and ESKAPE GroES/GroEL and identify significant differences in intra- and inter-ring cooperativity, required in the refolding cycle of client polypeptides. Previously, we observed that one-half of ESKAPE GroES/GroEL family members could not support cell viability when each was individually expressed in GroES/GroEL-deficient E. coli cells. Cell viability was found to be dependent on the allosteric compatibility between ESKAPE and E. coli subunits within mixed (E. coli and ESKAPE) tetradecameric GroEL complexes. Interestingly, differences in allostery did not necessarily result in differences in refolding rate for a given homotetradecameric chaperonin. Characterization of ESKAPE GroEL allostery, ATPase, and refolding rates in this study will serve to inform future studies focused on inhibitor design and mechanism of action studies.

Discovery of an eIF4A Inhibitor with a Novel Mechanism of Action.

Increased protein synthesis is a requirement for malignant growth, and as a result, translation has become a pharmaceutical target for cancer. The initiation of cap-dependent translation is enzymatically driven by the eukaryotic initiation factor (eIF)4A, an ATP-powered DEAD-box RNA-helicase that unwinds the messenger RNA secondary structure upstream of the start codon, enabling translation of downstream genes. A screen for inhibitors of eIF4A ATPase activity produced an intriguing hit that, surprisingly, was not ATP-competitive. A medicinal chemistry campaign produced the novel eIF4A inhibitor 28, which decreased BJAB Burkitt lymphoma cell viability. Biochemical and cellular studies, molecular docking, and functional assays uncovered that 28 is an RNA-competitive, ATP-uncompetitive inhibitor that engages a novel pocket in the RNA groove of eIF4A and inhibits unwinding activity by interfering with proper RNA binding and suppressing ATP hydrolysis. Inhibition of eIF4A through this unique mechanism may offer new strategies for targeting this promising intersection point of many oncogenic pathways.

A two-step resin based approach to reveal survivin-selective fluorescent probes.

The identification of modulators for proteins without assayable biochemical activity remains a challenge in chemical biology. The presented approach adapts a high-throughput fluorescence binding assay and functional chromatography, two protein-resin technologies, enabling the discovery and isolation of fluorescent natural product probes that target proteins independently of biochemical function. The resulting probes also suggest targetable pockets for lead discovery. Using human survivin as a model, we demonstrate this method with the discovery of members of the prodiginine family as fluorescent probes to the cancer target survivin.

Function, Therapeutic Potential, and Inhibition of Hsp70 Chaperones.

Hsp70s are among the most highly conserved proteins in all of biology. Through an iterative binding and release of exposed hydrophobic residues on client proteins, Hsp70s can prevent aggregation and promote folding to the native state of their client proteins. The human proteome contains eight canonical Hsp70s. Because Hsp70s are relatively promiscuous they play a role in folding a large proportion of the proteome. Hsp70s are implicated in disease through their ability to regulate protein homeostasis. In recent years, researchers have attempted to develop selective inhibitors of Hsp70 isoforms to better understand the role of individual isoforms in biology and as potential therapeutics. Selective inhibitors have come from rational design, forced localization, and serendipity, but the development of completely selective inhibitors remains elusive. In the present review, we discuss the Hsp70 structure and function, the known Hsp70 client proteins, the role of Hsp70s in disease, and current efforts to discover Hsp70 modulators.

Functional Differences between E. coli and ESKAPE Pathogen GroES/GroEL.

As the GroES/GroEL chaperonin system is the only bacterial chaperone that is essential under all conditions, we have been interested in the development of GroES/GroEL inhibitors as potential antibiotics. Using Escherichia coli GroES/GroEL as a surrogate, we have discovered several classes of GroES/GroEL inhibitors that show potent antibacterial activity against both Gram-positive and Gram-negative bacteria. However, it remains unknown if E. coli GroES/GroEL is functionally identical to other GroES/GroEL chaperonins and hence if our inhibitors will function against other chaperonins. Herein we report our initial efforts to characterize the GroES/GroEL chaperonins from clinically significant ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species). We used complementation experiments in GroES/GroEL-deficient and -null E. coli strains to report on exogenous ESKAPE chaperone function. In GroES/GroEL-deficient (but not knocked-out) E. coli, we found that only a subset of the ESKAPE GroES/GroEL chaperone systems could complement to produce a viable organism. Surprisingly, GroES/GroEL chaperone systems from two of the ESKAPE pathogens were found to complement in E. coli, but only in the strict absence of either E. coli GroEL (P. aeruginosa) or both E. coli GroES and GroEL (E. faecium). In addition, GroES/GroEL from S. aureus was unable to complement E. coli GroES/GroEL under all conditions. The resulting viable strains, in which E. coligroESL was replaced with ESKAPE groESL, demonstrated similar growth kinetics to wild-type E. coli, but displayed an elongated phenotype (potentially indicating compromised GroEL function) at some temperatures. These results suggest functional differences between GroES/GroEL chaperonins despite high conservation of amino acid identity.IMPORTANCE The GroES/GroEL chaperonin from E. coli has long served as the model system for other chaperonins. This assumption seemed valid because of the high conservation between the chaperonins. It was, therefore, shocking to discover ESKAPE pathogen GroES/GroEL formed mixed-complex chaperonins in the presence of E. coli GroES/GroEL, leading to loss of organism viability in some cases. Complete replacement of E. coligroESL with ESKAPE groESL restored organism viability, but produced an elongated phenotype, suggesting differences in chaperonin function, including client specificity and/or refolding cycle rates. These data offer important mechanistic insight into these remarkable machines, and the new strains developed allow for the synthesis of homogeneous chaperonins for biochemical studies and to further our efforts to develop chaperonin-targeted antibiotics.

A one-step, atom economical synthesis of thieno[2,3-d]pyrimidin-4-amine derivatives via a four-component reaction.

A Na2HPO4-catalyzed four-component reaction between a ketone, malononitrile, S8 and formamide has been realized for the first time. This reaction provides a concise approach to thieno[2,3-d]pyrimidin-4-amines, previously requiring 5 steps. The utility of this reaction was validated by preparing a multi-targeted kinase inhibitor and an inhibitor of the NRF2 pathway with excellent atom- and step-economy.

One-Step Synthesis of Thieno[2,3-d]pyrimidin-4(3H)-ones via a Catalytic Four-Component Reaction of Ketones, Ethyl Cyanoacetate, S8 and Formamide.

Thieno[2,3-d]pyrimidin-4(3H)-ones are important pharmacophores that previously required a three step synthesis with two chromatography steps. We herein report a green approach to the synthesis of this pharmacologically important class of compounds via a catalytic four-component reaction using a ketone, ethyl cyanoacetate, S8 and formamide. The reported reaction is characterized by step economy, reduced catalyst loading and easy purification.

An Isoform-Selective PTP1B Inhibitor Derived from Nitrogen-Atom Augmentation of Radicicol.

A library of natural products and their derivatives was screened for inhibition of protein tyrosine phosphatase (PTP) 1B, which is a validated drug target for the treatment of obesity and type II diabetes. Of those active in the preliminary assay, the most promising was compound 2 containing a novel pyrrolopyrazoloisoquinolone scaffold derived by treating radicicol (1) with hydrazine. This nitrogen-atom augmented radicicol derivative was found to be PTP1B selective relative to other highly homologous nonreceptor PTPs. Biochemical evaluation, molecular docking, and mutagenesis revealed 2 to be an allosteric inhibitor of PTP1B with a submicromolar Ki. Cellular analyses using C2C12 myoblasts indicated that 2 restored insulin signaling and increased glucose uptake.

A high throughput substrate binding assay reveals hexachlorophene as an inhibitor of the ER-resident HSP70 chaperone GRP78.

Glucose-regulated protein 78 (GRP78) is the ER resident 70 kDa heat shock protein 70 (HSP70) and has been hypothesized to be a therapeutic target for various forms of cancer due to its role in mitigating proteotoxic stress in the ER, its elevated expression in some cancers, and the correlation between high levels for GRP78 and a poor prognosis. Herein we report the development and use of a high throughput fluorescence polarization-based peptide binding assay as an initial step toward the discovery and development of GRP78 inhibitors. This assay was used in a pilot screen to discover the anti-infective agent, hexachlorophene, as an inhibitor of GRP78. Through biochemical characterization we show that hexachlorophene is a competitive inhibitor of the GRP78-peptide interaction. Biological investigations showed that this molecule induces the unfolded protein response, induces autophagy, and leads to apoptosis in a colon carcinoma cell model, which is known to be sensitive to GRP78 inhibition.

Hydroxybiphenylamide GroEL/ES Inhibitors Are Potent Antibacterials against Planktonic and Biofilm Forms of Staphylococcus aureus.

We recently reported the identification of a GroEL/ES inhibitor (1, N-(4-(benzo[ d]thiazol-2-ylthio)-3-chlorophenyl)-3,5-dibromo-2-hydroxybenzamide) that exhibited in vitro antibacterial effects against Staphylococcus aureus comparable to vancomycin, an antibiotic of last resort. To follow up, we have synthesized 43 compound 1 analogs to determine the most effective functional groups of the scaffold for inhibiting GroEL/ES and killing bacteria. Our results identified that the benzothiazole and hydroxyl groups are important for inhibiting GroEL/ES-mediated folding functions, with the hydroxyl essential for antibacterial effects. Several analogs exhibited >50-fold selectivity indices between antibacterial efficacy and cytotoxicity to human liver and kidney cells in cell culture. We found that MRSA was not able to easily generate acute resistance to lead inhibitors in a gain-of-resistance assay and that lead inhibitors were able to permeate through established S. aureus biofilms and maintain their bactericidal effects.

Sulfonamido-2-arylbenzoxazole GroEL/ES Inhibitors as Potent Antibacterials against Methicillin-Resistant Staphylococcus aureus (MRSA).

Extending from a study we recently published examining the antitrypanosomal effects of a series of GroEL/ES inhibitors based on a pseudosymmetrical bis-sulfonamido-2-phenylbenzoxazole scaffold, here, we report the antibiotic effects of asymmetric analogs of this scaffold against a panel of bacteria known as the ESKAPE pathogens ( Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species). While GroEL/ES inhibitors were largely ineffective against K. pneumoniae, A. baumannii, P. aeruginosa, and E. cloacae (Gram-negative bacteria), many analogs were potent inhibitors of E. faecium and S. aureus proliferation (Gram-positive bacteria, EC50 values of the most potent analogs were in the 1-2 µM range). Furthermore, even though some compounds inhibit human HSP60/10 biochemical functions in vitro (IC50 values in the 1-10 µM range), many of these exhibited moderate to low cytotoxicity to human liver and kidney cells (CC50 values > 20 µM).

A Chemical Biology Approach to Model Pontocerebellar Hypoplasia Type 1B (PCH1B).

Mutations of EXOSC3 have been linked to the rare neurological disorder known as Pontocerebellar Hypoplasia type 1B (PCH1B). EXOSC3 is one of three putative RNA-binding structural cap proteins that guide RNA into the RNA exosome, the cellular machinery that degrades RNA. Using RNAcompete, we identified a G-rich RNA motif binding to EXOSC3. Surface plasmon resonance (SPR) and microscale thermophoresis (MST) indicated an affinity in the low micromolar range of EXOSC3 for long and short G-rich RNA sequences. Although several PCH1B-causing mutations in EXOSC3 did not engage a specific RNA motif as shown by RNAcompete, they exhibited lower binding affinity to G-rich RNA as demonstrated by MST. To test the hypothesis that modification of the RNA-protein interface in EXOSC3 mutants may be phenocopied by small molecules, we performed an in-silico screen of 50 000 small molecules and used enzyme-linked immunosorbant assays (ELISAs) and MST to assess the ability of the molecules to inhibit RNA-binding by EXOSC3. We identified a small molecule, EXOSC3-RNA disrupting (ERD) compound 3 (ERD03), which ( i) bound specifically to EXOSC3 in saturation transfer difference nuclear magnetic resonance (STD-NMR), ( ii) disrupted the EXOSC3-RNA interaction in a concentration-dependent manner, and ( iii) produced a PCH1B-like phenotype with a 50% reduction in the cerebellum and an abnormally curved spine in zebrafish embryos. This compound also induced modification of zebrafish RNA expression levels similar to that observed with a morpholino against EXOSC3. To our knowledge, this is the first example of a small molecule obtained by rational design that models the abnormal developmental effects of a neurodegenerative disease in a whole organism.

Target-Based Screening against eIF4A1 Reveals the Marine Natural Product Elatol as a Novel Inhibitor of Translation Initiation with In Vivo Antitumor Activity.

Purpose: The DEAD-box RNA helicase eIF4A1 carries out the key enzymatic step of cap-dependent translation initiation and is a well-established target for cancer therapy, but no drug against it has entered evaluation in patients. We identified and characterized a natural compound with broad antitumor activities that emerged from the first target-based screen to identify novel eIF4A1 inhibitors.Experimental Design: We tested potency and specificity of the marine compound elatol versus eIF4A1 ATPase activity. We also assessed eIF4A1 helicase inhibition, binding between the compound and the target including binding site mutagenesis, and extensive mechanistic studies in cells. Finally, we determined maximum tolerated dosing in vivo and assessed activity against xenografted tumors.Results: We found elatol is a specific inhibitor of ATP hydrolysis by eIF4A1 in vitro with broad activity against multiple tumor types. The compound inhibits eIF4A1 helicase activity and binds the target with unexpected 2:1 stoichiometry at key sites in its helicase core. Sensitive tumor cells suffer acute loss of translationally regulated proteins, leading to growth arrest and apoptosis. In contrast to other eIF4A1 inhibitors, elatol induces markers of an integrated stress response, likely an off-target effect, but these effects do not mediate its cytotoxic activities. Elatol is less potent in vitro than the well-studied eIF4A1 inhibitor silvestrol but is tolerated in vivo at approximately 100× relative dosing, leading to significant activity against lymphoma xenografts.Conclusions: Elatol's identification as an eIF4A1 inhibitor with in vivo antitumor activities provides proof of principle for target-based screening against this highly promising target for cancer therapy. Clin Cancer Res; 24(17); 4256-70. ©2018 AACR.