Differential scanning fluorimetry (DSF) screen to identify inhibitors of Hsp60 protein-protein interactions.

There are relatively few methods available for discovering inhibitors of the protein-protein interactions (PPIs) that hold together homo-oligomers. We envisioned that Differential Scanning Fluorimetry (DSF) might be a versatile way to discover this type of inhibitor because oligomers are often more thermally stable than monomers. Using the homo-heptameric chaperonin, Hsp60, as a model, we screened ∼5000 diverse compounds in 384-well plates by DSF, revealing molecules that partially inhibited oligomerization. Because DSF does not require protein labeling or structural information, we propose that it could be a versatile way to uncover PPI inhibitors.

Predicting protein targets for drug-like compounds using transcriptomics.

An expanded chemical space is essential for improved identification of small molecules for emerging therapeutic targets. However, the identification of targets for novel compounds is biased towards the synthesis of known scaffolds that bind familiar protein families, limiting the exploration of chemical space. To change this paradigm, we validated a new pipeline that identifies small molecule-protein interactions and works even for compounds lacking similarity to known drugs. Based on differential mRNA profiles in multiple cell types exposed to drugs and in which gene knockdowns (KD) were conducted, we showed that drugs induce gene regulatory networks that correlate with those produced after silencing protein-coding genes. Next, we applied supervised machine learning to exploit drug-KD signature correlations and enriched our predictions using an orthogonal structure-based screen. As a proof-of-principle for this regimen, top-10/top-100 target prediction accuracies of 26% and 41%, respectively, were achieved on a validation of set 152 FDA-approved drugs and 3104 potential targets. We then predicted targets for 1680 compounds and validated chemical interactors with four targets that have proven difficult to chemically modulate, including non-covalent inhibitors of HRAS and KRAS. Importantly, drug-target interactions manifest as gene expression correlations between drug treatment and both target gene KD and KD of genes that act up- or down-stream of the target, even for relatively weak binders. These correlations provide new insights on the cellular response of disrupting protein interactions and highlight the complex genetic phenotypes of drug treatment. With further refinement, our pipeline may accelerate the identification and development of novel chemical classes by screening compound-target interactions.

High-throughput screen for inhibitors of protein-protein interactions in a reconstituted heat shock protein 70 (Hsp70) complex.

Protein-protein interactions (PPIs) are an important category of putative drug targets. Improvements in high-throughput screening (HTS) have significantly accelerated the discovery of inhibitors for some categories of PPIs. However, methods suitable for screening multiprotein complexes (e.g. those composed of three or more different components) have been slower to emerge. Here, we explored an approach that uses reconstituted multiprotein complexes (RMPCs). As a model system, we chose heat shock protein 70 (Hsp70), which is an ATP-dependent molecular chaperone that interacts with co-chaperones, including DnaJA2 and BAG2. The PPIs between Hsp70 and its co-chaperones stimulate nucleotide cycling. Thus, to re-create this ternary protein system, we combined purified human Hsp70 with DnaJA2 and BAG2 and then screened 100,000 diverse compounds for those that inhibited co-chaperone-stimulated ATPase activity. This HTS campaign yielded two compounds with promising inhibitory activity. Interestingly, one inhibited the PPI between Hsp70 and DnaJA2, whereas the other seemed to inhibit the Hsp70-BAG2 complex. Using secondary assays, we found that both compounds inhibited the PPIs through binding to allosteric sites on Hsp70, but neither affected Hsp70's intrinsic ATPase activity. Our RMPC approach expands the toolbox of biochemical HTS methods available for studying difficult-to-target PPIs in multiprotein complexes. The results may also provide a starting point for new chemical probes of the Hsp70 system.

Unbiased screen identifies aripiprazole as a modulator of abundance of the polyglutamine disease protein, ataxin-3.

No disease-modifying treatment exists for the fatal neurodegenerative polyglutamine disease known both as Machado-Joseph disease and spinocerebellar ataxia type 3. As a potential route to therapy, we identified small molecules that reduce levels of the mutant disease protein, ATXN3. Screens of a small molecule collection, including 1250 Food and Drug Administration-approved drugs, in a novel cell-based assay, followed by secondary screens in brain slice cultures from transgenic mice expressing the human disease gene, identified the atypical antipsychotic aripiprazole as one of the hits. Aripiprazole increased longevity in a Drosophila model of Machado-Joseph disease and effectively reduced aggregated ATXN3 species in flies and in brains of transgenic mice treated for 10 days. The aripiprazole-mediated decrease in ATXN3 abundance may reflect a complex response culminating in the modulation of specific components of cellular protein homeostasis. Aripiprazole represents a potentially promising therapeutic drug for Machado-Joseph disease and possibly other neurological proteinopathies.

Development of a capillary electrophoresis platform for identifying inhibitors of protein-protein interactions.

Methods for identifying chemical inhibitors of protein-protein interactions (PPIs) are often prone to discovery of false positives, particularly those caused by molecules that induce protein aggregation. Thus, there is interest in developing new platforms that might allow earlier identification of these problematic compounds. Capillary electrophoresis (CE) has been evaluated as a method to screen for PPI inhibitors using the challenging system of Hsp70 interacting with its co-chaperone Bag3. In the method, Hsp70 is labeled with a fluorophore, mixed with Bag3, and the resulting bound and free Hsp70 are separated and detected by CE with laser-induced fluorescence detection. The method used a chemically modified CE capillary to prevent protein adsorption. Inhibitors of the Hsp70-Bag3 interaction were detected by observing a reduction in the bound-to-free ratio. The method was used to screen a library of 3443 compounds, and the results were compared to those from a flow cytometry protein interaction assay. CE was found to produce a lower hit rate with more compounds that were reconfirmed in subsequent testing, suggesting greater specificity. This finding was attributed to the use of electropherograms to detect artifacts such as aggregators and to differences in protein modifications required to perform the different assays. Increases in throughput are required to make the CE method suitable for primary screens, but at the current stage of development it is attractive as a secondary screen to test hits found by higher-throughput methods.

Inhibitors of difficult protein-protein interactions identified by high-throughput screening of multiprotein complexes.

Protein-protein interactions (PPIs) are important in all aspects of cellular function, and there is interest in finding inhibitors of these contacts. However, PPIs with weak affinities and/or large interfaces have traditionally been more resistant to the discovery of inhibitors, partly because it is more challenging to develop high-throughput screening (HTS) methods that permit direct measurements of these physical interactions. Here, we explored whether the functional consequences of a weak PPI might be used as a surrogate for binding. As a model, we used the bacterial ATPase DnaK and its partners DnaJ and GrpE. Both DnaJ and GrpE bind DnaK and catalytically accelerate its ATP cycling, so we used stimulated nucleotide turnover to indirectly report on these PPIs. In pilot screens, we identified compounds that block activation of DnaK by either DnaJ or GrpE. Interestingly, at least one of these molecules blocked binding of DnaK to DnaJ, while another compound disrupted allostery between DnaK and GrpE without altering the physical interaction. These findings suggest that the activity of a reconstituted multiprotein complex might be used in some cases to identify allosteric inhibitors of challenging PPIs.

Synthesis and initial evaluation of YM-08, a blood-brain barrier permeable derivative of the heat shock protein 70 (Hsp70) inhibitor MKT-077, which reduces tau levels.

The molecular chaperone, heat shock protein 70 (Hsp70), is an emerging drug target for treating neurodegenerative tauopathies. We recently found that one promising Hsp70 inhibitor, MKT-077, reduces tau levels in cellular models. However, MKT-077 does not penetrate the blood-brain barrier (BBB), limiting its use as either a clinical candidate or probe for exploring Hsp70 as a drug target in the central nervous system (CNS). We hypothesized that replacing the cationic pyridinium moiety in MKT-077 with a neutral pyridine might improve its clogP and enhance its BBB penetrance. To test this idea, we designed and synthesized YM-08, a neutral analogue of MKT-077. Like the parent compound, YM-08 bound to Hsp70 in vitro and reduced phosphorylated tau levels in cultured brain slices. Pharmacokinetic evaluation in CD1 mice showed that YM-08 crossed the BBB and maintained a brain/plasma (B/P) value of ∼0.25 for at least 18 h. Together, these studies suggest that YM-08 is a promising scaffold for the development of Hsp70 inhibitors suitable for use in the CNS.

A screen for modulators of large T antigen's ATPase activity uncovers novel inhibitors of Simian Virus 40 and BK virus replication.

New polyomaviruses are continually being identified, and it is likely that links between this virus family and disease will continue to emerge. Unfortunately, a specific treatment for polyomavirus-associated disease is lacking. Because polyomaviruses express large Tumor Antigen, TAg, we hypothesized that small molecule inhibitors of the essential ATPase activity of TAg would inhibit viral replication. Using a new screening platform, we identified inhibitors of TAg's ATPase activity. Lead compounds were moved into a secondary assay, and ultimately two FDA approved compounds, bithionol and hexachlorophene, were identified as the most potent TAg inhibitors known to date. Both compounds inhibited Simian Virus 40 replication as assessed by plaque assay and quantitative PCR. Moreover, these compounds inhibited BK virus, which causes BKV Associated Nephropathy. In neither case was host cell viability compromised at these concentrations. Our data indicate that directed screening for TAg inhibitors is a viable method to identify polyomavirus inhibitors, and that bithionol and hexachlorophene represent lead compounds that may be further modified and/or ultimately used to combat diseases associated with polyomavirus infection.

Molecular chaperones and regulation of tau quality control: strategies for drug discovery in tauopathies.

Tau is a microtubule-associated protein that accumulates in at least 15 different neurodegenerative disorders, which are collectively referred to as tauopathies. In these diseases, tau is often hyperphosphorylated and found in aggregates, including paired helical filaments, neurofibrillary tangles and other abnormal oligomers. Tau aggregates are associated with neuron loss and cognitive decline, which suggests that this protein can somehow evade normal quality control allowing it to aberrantly accumulate and become proteotoxic. Consistent with this idea, recent studies have shown that molecular chaperones, such as heat shock protein 70 and heat shock protein 90, counteract tau accumulation and neurodegeneration in disease models. These molecular chaperones are major components of the protein quality control systems and they are specifically involved in the decision to retain or degrade many proteins, including tau and its modified variants. Thus, one potential way to treat tauopathies might be to either accelerate interactions of abnormal tau with these quality control factors or tip the balance of triage towards tau degradation. In this review, we summarize recent findings and suggest models for therapeutic intervention.

Chemical screens against a reconstituted multiprotein complex: myricetin blocks DnaJ regulation of DnaK through an allosteric mechanism.

DnaK is a molecular chaperone responsible for multiple aspects of bacterial proteostasis. The intrinsically slow ATPase activity of DnaK is stimulated by its co-chaperone, DnaJ, and these proteins often work in concert. To identify inhibitors we screened plant-derived extracts against a reconstituted mixture of DnaK and DnaJ. This approach resulted in the identification of flavonoids, including myricetin, which inhibited activity by up to 75%. Interestingly, myricetin prevented DnaJ-mediated stimulation of ATPase activity, with minimal impact on either DnaK's intrinsic turnover rate or its stimulation by another co-chaperone, GrpE. Using NMR, we found that myricetin binds DnaK at an unanticipated site between the IB and IIB subdomains and that it allosterically blocked binding of DnaK to DnaJ. Together, these results highlight a "gray box" screening approach, which might facilitate the identification of inhibitors of other protein-protein interactions.

Identification of small molecules that modify the protein folding activity of heat shock protein 70.

Molecular chaperones, such as heat shock protein 70 (Hsp70) and its bacterial ortholog DnaK, play numerous important roles in protein folding. In vitro, this activity can be observed by incubating purified chaperones with denatured substrates and measuring the recovery of properly folded protein. In an effort to rapidly identify small molecules that modify this folding activity, we modified an existing method for use in 96-well plates. In this assay, denatured firefly luciferase was treated with a mixture of DnaK and prospective chemical modulators. The luminescence of refolded luciferase was used to follow the reaction progress, and counterscreens excluded compounds that target luciferase; thus, hits from these screens modify protein folding via their effects on the function of the chaperone machine. Using this platform, we screened a pilot chemical library and found five new inhibitors of DnaK and one compound that promoted folding. These chemical probes may be useful in studies aimed at understanding the many varied roles of chaperones in cellular protein folding. Moreover, this assay provides the opportunity to rapidly screen for additional compounds that might regulate the folding activity of Hsp70.

Target identification for a promising anti-lupus drug.

Target identification is one of the more difficult aspects of modern chemical genetics. In this issue of Chemistry & Biology, Johnson et al. provide a compelling case for going through the trouble. As a byproduct of their identification of the cellular target of an anti-lupus compound, they have uncovered a powerful tool for the characterization of a fascinating molecular machine, the F1F0-ATP synthase.