Showing papers by "Jason E. Gestwicki published in 2018"

Mapping interactions with the chaperone network reveals factors that protect against tau aggregation.

Abstract: A network of molecular chaperones is known to bind proteins ('clients') and balance their folding, function and turnover. However, it is often unclear which chaperones are critical for selective recognition of individual clients. It is also not clear why these key chaperones might fail in protein-aggregation diseases. Here, we utilized human microtubule-associated protein tau (MAPT or tau) as a model client to survey interactions between ~30 purified chaperones and ~20 disease-associated tau variants (~600 combinations). From this large-scale analysis, we identified human DnaJA2 as an unexpected, but potent, inhibitor of tau aggregation. DnaJA2 levels were correlated with tau pathology in human brains, supporting the idea that it is an important regulator of tau homeostasis. Of note, we found that some disease-associated tau variants were relatively immune to interactions with chaperones, suggesting a model in which avoiding physical recognition by chaperone networks may contribute to disease.

SIRT1 Deacetylates Tau and Reduces Pathogenic Tau Spread in a Mouse Model of Tauopathy

Abstract: Hyperacetylation of tau has been implicated in neurodegeneration and cognitive decline in tauopathy brains. The nicotinamide adenosine dinucleotide-dependent class-III protein deacetylase SIRT1 is one of the major enzymes involved in removal of acetyl groups from tau in vitro However, whether SIRT1 regulates acetylation of pathogenic tau and ameliorates tau-mediated pathogenesis remains unclear. Here, we report deacetylating activity of SIRT1 for acetylated Lys174 (K174) of tau in tauP301S transgenic mice with a brain-specific SIRT1 deletion. We show that SIRT1 deficiency leads to exacerbation of premature mortality, synapse loss, and behavioral disinhibition in tauP301S transgenic mice of both sexes. By contrast, SIRT1 overexpression by stereotaxic delivery of adeno-associated virus that encodes SIRT1 into the hippocampus reduces acetylated K174 tau. Furthermore, SIRT1 overexpression significantly attenuates the spread of tau pathology into anatomically connected brain regions of tauP301S transgenic mice of both sexes. These findings suggest the functional importance of SIRT1 in regulating pathogenic tau acetylation and in suppressing the spread of tau pathology in vivoSIGNIFICANCE STATEMENT In neurodegenerative disorders with inclusions of microtubule-associated protein tau, aberrant lysine acetylation of tau plays critical roles in promoting tau accumulation and toxicity. Identifying strategies to deacetylate tau could interfere with disease progression; however, little is known about how pathogenic tau is deacetylated in vivo Here we show that the protein deacetylase SIRT1 reduces tau acetylation in a mouse model of neurodegeneration. SIRT1 deficiency in the brain aggravates synapse loss and behavioral disinhibition, and SIRT1 overexpression ameliorates propagation of tau pathology.

Targeting the Hsp40/Hsp70 Chaperone Axis as a Novel Strategy to Treat Castration-Resistant Prostate Cancer.

Abstract: Castration-resistant prostate cancer (CRPC) is characterized by reactivation of androgen receptor (AR) signaling, in part by elevated expression of AR splice variants (ARv) including ARv7, a constitutively active, ligand binding domain (LBD)-deficient variant whose expression has been correlated with therapeutic resistance and poor prognosis. In a screen to identify small-molecule dual inhibitors of both androgen-dependent and androgen-independent AR gene signatures, we identified the chalcone C86. Binding studies using purified proteins and CRPC cell lysates revealed C86 to interact with Hsp40. Pull-down studies using biotinylated-C86 found Hsp40 present in a multiprotein complex with full-length (FL-) AR, ARv7, and Hsp70 in CRPC cells. Treatment of CRPC cells with C86 or the allosteric Hsp70 inhibitor JG98 resulted in rapid protein destabilization of both FL-AR and ARv, including ARv7, concomitant with reduced FL-AR- and ARv7-mediated transcriptional activity. The glucocorticoid receptor, whose elevated expression in a subset of CRPC also leads to androgen-independent AR target gene transcription, was also destabilized by inhibition of Hsp40 or Hsp70. In vivo, Hsp40 or Hsp70 inhibition demonstrated single-agent and combinatorial activity in a 22Rv1 CRPC xenograft model. These data reveal that, in addition to recognized roles of Hsp40 and Hsp70 in FL-AR LBD remodeling, ARv lacking the LBD remain dependent on molecular chaperones for stability and function. Our findings highlight the feasibility and potential benefit of targeting the Hsp40/Hsp70 chaperone axis to treat prostate cancer that has become resistant to standard antiandrogen therapy.Significance: These findings highlight the feasibility of targeting the Hsp40/Hsp70 chaperone axis to treat CRPC that has become resistant to standard antiandrogen therapy. Cancer Res; 78(14); 4022-35. ©2018 AACR.

Exploration of Benzothiazole Rhodacyanines as Allosteric Inhibitors of Protein–Protein Interactions with Heat Shock Protein 70 (Hsp70)

Abstract: Cancer cells rely on the chaperone heat shock protein 70 (Hsp70) for survival and proliferation. Recently, benzothiazole rhodacyanines have been shown to bind an allosteric site on Hsp70, interrupting its binding to nucleotide-exchange factors (NEFs) and promoting cell death in breast cancer cell lines. However, proof-of-concept molecules, such as JG-98, have relatively modest potency (EC50 ≈ 0.7–0.4 μM) and are rapidly metabolized in animals. Here, we explored this chemical series through structure- and property-based design of ∼300 analogs, showing that the most potent had >10-fold improved EC50 values (∼0.05 to 0.03 μM) against two breast cancer cells. Biomarkers and whole genome CRISPRi screens confirmed members of the Hsp70 family as cellular targets. On the basis of these results, JG-231 was found to reduce tumor burden in an MDA-MB-231 xenograft model (4 mg/kg, ip). Together, these studies support the hypothesis that Hsp70 may be a promising target for anticancer therapeutics.

Competing protein-protein interactions regulate binding of Hsp27 to its client protein tau.

Abstract: Small heat shock proteins (sHSPs) are a class of oligomeric molecular chaperones that limit protein aggregation. However, it is often not clear where sHSPs bind on their client proteins or how these protein-protein interactions (PPIs) are regulated. Here, we map the PPIs between human Hsp27 and the microtubule-associated protein tau (MAPT/tau). We find that Hsp27 selectively recognizes two aggregation-prone regions of tau, using the conserved β4-β8 cleft of its alpha-crystallin domain. The β4-β8 region is also the site of Hsp27-Hsp27 interactions, suggesting that competitive PPIs may be an important regulatory paradigm. Indeed, we find that each of the individual PPIs are relatively weak and that competition for shared sites seems to control both client binding and Hsp27 oligomerization. These findings highlight the importance of multiple, competitive PPIs in the function of Hsp27 and suggest that the β4-β8 groove acts as a tunable sensor for clients.

Protein-Protein Interactions in the Molecular Chaperone Network.

Abstract: ConspectusMolecular chaperones play a central role in protein homeostasis (a.k.a. proteostasis) by balancing protein folding, quality control, and turnover. To perform these diverse tasks, chaperones need the malleability to bind nearly any “client” protein and the fidelity to detect when it is misfolded. Remarkably, these activities are carried out by only ∼180 dedicated chaperones in humans. How do a relatively small number of chaperones maintain cellular and organismal proteostasis for an entire proteome? Furthermore, once a chaperone binds a client, how does it “decide” what to do with it? One clue comes from observations that individual chaperones engage in protein–protein interactions (PPIs)—both with each other and with their clients. These physical links coordinate multiple chaperones into organized, functional complexes and facilitate the “handoff” of clients between them. PPIs also link chaperones and their clients to other cellular pathways, such as those that mediate trafficking (e.g., cytoske...

Myopathy associated BAG3 mutations lead to protein aggregation by stalling Hsp70 networks

Abstract: BAG3 is a multi-domain hub that connects two classes of chaperones, small heat shock proteins (sHSPs) via two isoleucine-proline-valine (IPV) motifs and Hsp70 via a BAG domain. Mutations in either the IPV or BAG domain of BAG3 cause a dominant form of myopathy, characterized by protein aggregation in both skeletal and cardiac muscle tissues. Surprisingly, for both disease mutants, impaired chaperone binding is not sufficient to explain disease phenotypes. Recombinant mutants are correctly folded, show unaffected Hsp70 binding but are impaired in stimulating Hsp70-dependent client processing. As a consequence, the mutant BAG3 proteins become the node for a dominant gain of function causing aggregation of itself, Hsp70, Hsp70 clients and tiered interactors within the BAG3 interactome. Importantly, genetic and pharmaceutical interference with Hsp70 binding completely reverses stress-induced protein aggregation for both BAG3 mutations. Thus, the gain of function effects of BAG3 mutants act as Achilles heel of the HSP70 machinery. BAG3 is a Hsp70 co-chaperone that is highly expressed in muscles. Here the authors show that several myofibrillar myopathy causing BAG3 mutations are not impaired in Hsp70 binding, but rather impair the ADP-ATP exchange step of the Hsp70 cycle, causing the aggregation of BAG3, Hsp70 and Hsp70 clients and leading to a collapse of protein homeostasis.

KHS101 disrupts energy metabolism in human glioblastoma cells and reduces tumor growth in mice

Abstract: Pharmacological inhibition of uncontrolled cell growth with small-molecule inhibitors is a potential strategy for treating glioblastoma multiforme (GBM), the most malignant primary brain cancer. We showed that the synthetic small-molecule KHS101 promoted tumor cell death in diverse GBM cell models, independent of their tumor subtype, and without affecting the viability of noncancerous brain cell lines. KHS101 exerted cytotoxic effects by disrupting the mitochondrial chaperone heat shock protein family D member 1 (HSPD1). In GBM cells, KHS101 promoted aggregation of proteins regulating mitochondrial integrity and energy metabolism. Mitochondrial bioenergetic capacity and glycolytic activity were selectively impaired in KHS101-treated GBM cells. In two intracranial patient-derived xenograft tumor models in mice, systemic administration of KHS101 reduced tumor growth and increased survival without discernible side effects. These findings suggest that targeting of HSPD1-dependent metabolic pathways might be an effective strategy for treating GBM.

Predicting protein targets for drug-like compounds using transcriptomics

Abstract: 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.

Heat Shock Protein 70 (Hsp70) Suppresses RIP1-Dependent Apoptotic and Necroptotic Cascades

Abstract: Hsp70 is a molecular chaperone that binds to “client” proteins and protects them from protein degradation. Hsp70 is essential for the survival of many cancer cells, but it is not yet clear which of its clients are involved. Using structurally distinct chemical inhibitors, we found that many of the well-known clients of the related chaperone, Hsp90, are not strikingly responsive to Hsp70 inhibition. Rather, Hsp70 appeared to be important for the stability of the RIP1 (RIPK1) regulators: cIAP1/2 (BIRC1 and BIRC3), XIAP, and cFLIP S/L (CFLAR). These results suggest that Hsp70 limits apoptosis and necroptosis pathways downstream of RIP1. Consistent with this model, MDA-MB-231 breast cancer cells treated with Hsp70 inhibitors underwent apoptosis, while cotreatment with z-VAD.fmk switched the cell death pathway to necroptosis. In addition, cell death in response to Hsp70 inhibitors was strongly suppressed by RIP1 knockdown or inhibitors. Thus, these data indicate that Hsp70 plays a previously unrecognized and important role in suppressing RIP1 activity. Implications: These findings clarify the role of Hsp70 in prosurvival signaling and suggest IAPs as potential new biomarkers for Hsp70 inhibition. Mol Cancer Res; 16(1); 58–68. ©2017 AACR .

HSC70 is a chaperone for wild-type and mutant cardiac myosin binding protein C

Abstract: Cardiac myosin binding protein C (MYBPC3) is the most commonly mutated gene associated with hypertrophic cardiomyopathy (HCM). Haploinsufficiency of full-length MYBPC3 and disruption of proteostasis have both been proposed as central to HCM disease pathogenesis. Discriminating the relative contributions of these 2 mechanisms requires fundamental knowledge of how turnover of WT and mutant MYBPC3 proteins is regulated. We expressed several disease-causing mutations in MYBPC3 in primary neonatal rat ventricular cardiomyocytes. In contrast to WT MYBPC3, mutant proteins showed reduced expression and failed to localize to the sarcomere. In an unbiased coimmunoprecipitation/mass spectrometry screen, we identified HSP70-family chaperones as interactors of both WT and mutant MYBPC3. Heat shock cognate 70 kDa (HSC70) was the most abundant chaperone interactor. Knockdown of HSC70 significantly slowed degradation of both WT and mutant MYBPC3, while pharmacologic activation of HSC70 and HSP70 accelerated degradation. HSC70 was expressed in discrete striations in the sarcomere. Expression of mutant MYBPC3 did not affect HSC70 localization, nor did it induce a protein folding stress response or ubiquitin proteasome dysfunction. Together these data suggest that WT and mutant MYBPC3 proteins are clients for HSC70, and that the HSC70 chaperone system plays a major role in regulating MYBPC3 protein turnover.

Therapeutic Strategies for Restoring Tau Homeostasis.

Abstract: Normal tau homeostasis is achieved when the synthesis, processing, and degradation of the protein is balanced. Together, the pathways that regulate tau homeostasis ensure that the protein is at the proper levels and that its posttranslational modifications and subcellular localization are appropriately controlled. These pathways include the enzymes responsible for posttranslational modifications, those systems that regulate mRNA splicing, and the molecular chaperones that control tau turnover and its binding to microtubules. In tauopathies, this delicate balance is disturbed. Tau becomes abnormally modified by posttranslational modification, it loses affinity for microtubules, and it accumulates in proteotoxic aggregates. How and why does this imbalance occur? In this review, we discuss how molecular chaperones and other components of the protein homeostasis (e.g., proteostasis) network normally govern tau quality control. We also discuss how aging might reduce the capacity of these systems and how tau mutations might further affect this balance. Finally, we discuss how small-molecule inhibitors are being used to probe and perturb the tau quality-control systems, playing a particularly prominent role in revealing the logic of tau homeostasis. As such, there is now interest in developing these chemical probes into therapeutics, with the goal of restoring normal tau homeostasis to treat disease.

A Local Allosteric Network in Heat Shock Protein 70 (Hsp70) Links Inhibitor Binding to Enzyme Activity and Distal Protein-Protein Interactions.

Abstract: Allosteric inhibitors can be more difficult to optimize without an understanding of how their binding influences the conformational motions of the target. Here, we used an integrated computational and experimental approach to probe the molecular mechanism of an allosteric inhibitor of heat shock protein 70 (Hsp70). The anticancer compound, MKT-077, is known to bind a conserved site in members of the Hsp70 family, which favors the ADP-bound state and interferes with a protein-protein interaction (PPI) at long range. However, the binding site does not overlap with either the nucleotide-binding cleft or the PPI contact surface, so its mechanism is unclear. To this end, we modeled Hsp70's internal dynamics and studied how MKT-077 alters local sampling of its allosteric states. The results pointed to a set of concerted motions between five loops in Hsp70's nucleotide-binding domain (NBD), surrounding the MKT-077 binding site. To test this prediction, we mutated key residues and monitored chaperone activities in vitro. Together, the results indicate that MKT-077 interacts with loop222 to favor a pseudo-ADP bound conformer of Hsp70's NBD, even when ATP is present. We used this knowledge to synthesize an analog of MKT-077 that would better prevent motions of loop222 and confirmed that it had improved antiproliferative activity in breast cancer cells. These results provide an example of how to unlock and leverage the complex mechanisms of allosteric inhibitors.

Protein cross-linking capillary electrophoresis at increased throughput for a range of protein–protein interactions

Abstract: Tools for measuring affinities and stoichiometries of protein–protein complexes are valuable for elucidating the role of protein–protein interactions (PPIs) in governing cell functions and screening for PPI modulators. Such measurements can be challenging because PPIs can span a wide range of affinities and include stoichiometries from dimers to high order oligomers. Also, most techniques require large amounts of protein which can hamper research for difficult to obtain proteins. Protein cross-linking capillary electrophoresis (PXCE) has the potential to directly measure PPIs and even resolve multiple PPIs while consuming attomole quantities. Previously PXCE has only been used for high affinity, 1 : 1 complexes; here we expand the utility of PXCE to access a wide range of PPIs including weak and multimeric oligomers. Use of glutaraldehyde as the cross-linking agent was key to advancing the method because of its rapid reaction kinetics. A 10 s reaction time was found to be sufficient for cross-linking and quantification of seven different PPIs with Kd values ranging from low μM to low nM including heat shock protein 70 (Hsp70) interacting with heat shock organizing protein (3.8 ± 0.7 μM) and bcl2 associated anthanogene (26 ± 6 nM). Non-specific cross-linking of protein aggregates was found to be minimal at protein concentrations <20 μM as assessed by size exclusion chromatography. PXCE was sensitive enough to measure changes in PPI affinity induced by the protein nucleotide state or point mutations in the protein-binding site. Further, several interactions could be resolved in a single run, including Hsp70 monomer, homodimer and Hsp70 complexed the with c-terminus of Hsp70 interacting protein (CHIP). Finally, the throughput of PXCE was increased to 1 min per sample suggesting potential for utility in screening.

Tau Secretion and Propagation Is Regulated by p300/CBP via Autophagy-Lysosomal Pathway in Tauopathy

Abstract: SUMMARY The trans-neuronal propagation of tau has been implicated in the progression of tau-mediated neurodegeneration. Tau secretion from neurons is the first step in tau transmission, but little is known about the cellular mechanism. Here, we report that p300/CBP, the lysine acetyltransferase that acetylates tau and regulates its homeostasis and toxicity, serves as a key regulator of tau secretion by inhibiting the autophagy-lysosomal pathway (ALP). Increased p300/CBP activity was associated with impaired function of this pathway in a tau transgenic mouse model. p300/CBP hyperactivation increased tau secretion by blocking autophagic flux. Conversely, inhibiting p300/CBP genetically or pharmacologically promoted autophagic flux, and reduced tau accumulation, tau secretion, and tau propagation in fibril-induced tau spreading models in vitro and in vivo. Our findings show that p300/CBP-induced impairment in the ALP underlies excessive unconventional secretion and pathogenic spread of tau.

Managing the Spatial Covariance of Genetic Diversity in Niemann-Pick C1 Through Modulation of the Hsp70 Chaperone System

Abstract: Genetic diversity provides a rich repository for understanding the role of proteostasis in the management of the protein fold to allow biology to evolve through variation in the population and in response to the environment. Failure in proteostasis can trigger multiple disease states affecting both human health and lifespan. Niemann-Pick C (NPC) disease is a genetic disorder mainly caused by mutations in NPC1, a multi-spanning transmembrane protein that is trafficked through the exocytic pathway to late endosomes and lysosomes (LE/Ly) to manage cholesterol homeostasis. Proteostatic defects triggered by >600 NPC1 variants found in the human population inhibit export of NPC1 protein from ER or function in downstream LE/Ly, leading to accumulation of cholesterol and rapid onset neurodegeneration in childhood for most patients. We now show that chemical allosteric inhibitors, such as JG98, targeting the cytosolic Hsp70 chaperone/co-chaperone complex improves the trafficking and stability of NPC1 variants with diverse NPC1 genotypes. By exploiting the knowledge-base of NPC1 variants found in the world-wide patient population using Variation Spatial Profiling (VSP), a Gaussian-process based machine learning (ML) approach, we show how the Hsp70 chaperone system alters the spatial covariance (SCV) tolerance of the ER and the SCV set-points for each residue of the NPC1 polypeptide chain differentially to improve trafficking efficiency and post-ER stability for variants distributed across the entire NPC1 polypeptide. The impact of JG98 is supported by the observation that silencing of Hsp70 specific nucleotide exchange factors (NEF) (BCL-anthogene (BAG) family) co-chaperones significantly improve the folding status of NPC1 variants. Together, these studies suggest that targeting the cytosolic Hsp70 system to adjust the SCV tolerance of the proteostasis network can improve recognition of the plasticity of the NPC1 fold found in the disease population for trafficking to the LE/Ly compartments.

Management of Hsp90-Dependent Protein Folding by Small Molecule Targeting the Aha1 Co-Chaperone

Abstract: The core cytosolic Hsp90 chaperone/co-chaperone complex plays a critical role in proteostasis management of human health and disease To identify novel compounds that alter the ability of the Hsp90 co-chaperone Aha1 to modulate the ATPase activity found in multiple folding diseases ranging from steroid hormone receptor (SHR) sensitive prostate cancer to tauopathies associated with neurodegenerative diseases, we employed a high throughput screening (HTS) assay to monitor selectively Aha1-stimulated Hsp90 (ASH) ATPase activity The ASH assay identified SEW04784 (SEW), a small molecule that disrupts ASH activity without inhibiting the basal Hsp90 ATPase activity NMR analysis reveals that SEW binds to the C-terminal domain of Aha1 to disrupt its asymmetric binding to Hsp90 leading to abrogation of its chaperoning activity of Hsp90 SEW exhibits therapeutic potential by blocking the transcriptional activity of prostate cancer (PCa) associated variants of the androgen receptor (AR) in a cell-based model of PCa Additionally, SEW exhibits the ability to clear toxic, phosphorylated tau aggregated species associated with tauopathies By not directly impacting the basal ATPase function of the abundant and ubiquitous Hsp90, SEW could provide a therapeutic approach for mitigation of client-specific proteostatic disease

Predicting protein targets for drug-like compounds using transcriptomics

Abstract: The development of an expanded chemical space for screening is an essential step in the challenge of identifying chemical probes for new, genomic-era protein targets. However, the difficulty of identifying targets for novel compounds leads to the prioritization of synthesis linked to known active scaffolds that bind familiar protein families, slowing the exploration of available chemical space. To change this paradigm, we validated a new pipeline capable of identifying compound-protein interactions even for compounds with no similarity to known drugs. Based on differential mRNA profiles from drug treatments and gene knockdowns across multiple cell types, we show that drugs cause gene regulatory network effects that correlate with those produced by silencing their target protein-coding gene. Applying supervised machine learning to exploit compound-knockdown signature correlations and enriching our predictions using an orthogonal structure-based screen, we achieved top-10/top-100 target prediction accuracies of 26%/41%, respectively, on a validation set 152 FDA-approved drugs and 3104 potential targets. We further predicted targets for 1680 compounds and validated a total of seven novel interactions with four difficult targets, including non-covalent modulators of HRAS and KRAS. We found that drug-target interactions manifest as gene expression correlations between drug treatment and both target gene knockdown and up/down-stream knockdowns. These correlations provide biologically relevant insight on the cell-level impact of disrupting protein interactions, highlighting the complex genetic phenotypes of drug treatments. Our pipeline can accelerate the identification and development of novel chemistries with potential to become drugs by screening for compound-target interactions in the full human interactome.

Proteome-scale detection of drug-target interactions using correlations in transcriptomic perturbations

Abstract: Systems biology seeks to understand how normal and disease protein networks respond when specific interactions are disrupted. A first step towards this goal is identifying the molecular target(s) of bioactive compounds. Here, we hypothesize that inhibitory drugs should produce network-level effects similar to silencing the inhibited gene and show that drug-protein interactions are encoded in mRNA expression profile correlations. We use machine learning to classify correlations between drug- and knockdown-induced expression signatures and enrich our predictions through a structure-based screen. Interactions manifest both as direct correlations between drug and target knockdowns, and as indirect correlations with up/downstream knockdowns. Cross-validation on 152 FDA-approved drugs and 3104 potential targets achieved top 10/100 prediction accuracies of 26/41%. We apply our method to 1680 bioactive compounds and experimentally validate five previously unknown interactions. Our pipeline can accelerate drug discovery by matching existing compounds to new therapeutic targets while informing on network and multi-target effects.