Protein-protein Interaction Inhibitors Research Articles

Electrophiles and Keap1-Nrf2 protein-protein interaction inhibitors for prevention and treatment of chronic disease

Electrophiles and Keap1-Nrf2 protein-protein interaction inhibitors for prevention and treatment of chronic disease

Innovative peptide architectures: advancements in foldamers and stapled peptides for drug discovery.

Peptide foldamers play a critical role in pharmaceutical research and biomedical applications. This review highlights recent (post-2020) advancements in novel foldamers, synthetic techniques, and their applications in pharmaceutical research. The authors summarize the structures and applications of peptide foldamers such as α, β, γ-peptides, hydrocarbon-stapled peptides, urea-type foldamers, sulfonic-γ-amino acid foldamers, aromatic foldamers, and peptoids, which tackle the challenges of traditional peptide drugs. Regarding antimicrobial use, foldamers have shown progress in their potential against drug-resistant bacteria. In drug development, peptide foldamers have been used as drug delivery systems (DDS) and protein-protein interaction (PPI) inhibitors. These structures exhibit resistance to enzymatic degradation, are promising for therapeutic delivery, and disrupt crucial PPIs associated with diseases such as cancer with specificity, versatility, and stability, which are useful therapeutic properties. However, the complexity and cost of their synthesis, along with the necessity for thorough safety and efficacy assessments, necessitate extensive research and cross-sector collaboration. Advances in synthesis methods, computational modeling, and targeted delivery systems are essential for fully realizing the therapeutic potential of foldamers and integrating them into mainstream medical treatments.

α-Hydrazino Acid Insertion Governs Peptide Organization in Solution by Local Structure Ordering.

In this work, we have applied the concept of α-hydrazino acid insertion in a peptide sequence as a means of structurally organizing a potential protein-protein interactions (PPI) inhibitor. Hydrazino peptides characterized by the incorporation of an α-hydrazino acid at specific positions introduce an additional nitrogen atom into their backbone. This modification leads to a change in the electrostatic properties of the peptide and induces the restructuring of its hydrogen bonding network, resulting in conformational changes toward more stable structural motifs. Despite the successful use of synthetic hydrazino oligomers in binding to nucleic acids, the structural changes due to the incorporation of α-hydrazino acid into short natural peptides in solution are still poorly understood. Based on NMR data, we report structural models of p53-derived hydrazino peptides with elements of localized peptide structuring in the form of an α-, β-, or γ-turn as a result of hydrazino modification in the peptide backbone. The modifications could potentially lead to the preorganization of a helical secondary peptide structure in a solution that is favorable for binding to a biological receptor. Spectroscopically, we observed that the ensemble averaged rapidly interconverting conformations, including isomerization of the E-Z hydrazide bond. This further increases the adaptability by expanding the conformational space of hydrazine peptides as potential protein-protein interaction antagonists.

Validation of aminodeoxychorismate synthase and anthranilate synthase as novel targets for bispecific antibiotics inhibiting conserved protein-protein interactions.

Antibiotics are indispensable for the treatment of bacterial infections in human and veterinary medicine and are thus a major pillar of modern medicine. However, the exposure of bacteria to antibiotics generates an unintentional selective pressure on bacterial assemblies that over time promotes the development or acquisition of resistance mechanisms, allowing pathogens to escape the treatment. In that manner, humanity is in an ever-lasting race with pathogens to come up with new treatment options before resistances emerge. In general, antibiotics with novel modes of action require more complex pathogen adaptations as compared to chemical derivates of existing entities, thus delaying the emergence of resistance. In this contribution, we use modified Escherichia coli strains to validate two novel targets required for folate and tryptophan biosynthesis that can potentially be targeted by one and the same bispecific protein-protein interaction inhibitor and promise increased robustness against bacterial resistances.

Verteporfin inhibits TGF-β signaling by disrupting the Smad2/3-Smad4 interaction.

Transforming growth factor-β (TGF-β) signaling plays a crucial role in pathogenesis, such as accelerating tissue fibrosis and promoting tumor development at the later stages of tumorigenesis by promoting epithelial-mesenchymal transition (EMT), cancer cell migration, and invasion. Targeting TGF-β signaling is a promising therapeutic approach, but nonspecific inhibition may result in adverse effects. In this study, we focus on the Smad2/3-Smad4 complex, a key component in TGF-β signaling transduction, as a potential target for cancer therapy. Through a phase-separated condensate-aided biomolecular interaction system, we identified verteporfin (VP) as a small-molecule inhibitor that specifically targets the Smad2/3-Smad4 interaction. VP effectively disrupted the interaction between Smad2/3 and Smad4 and thereby inhibited canonical TGF-β signaling, but not the interaction between Smad1 and Smad4 in bone morphogenetic protein (BMP) signaling. Furthermore, VP exhibited inhibitory effects on TGF-β-induced EMT and cell migration. Our findings indicate a novel approach to develop protein-protein interaction inhibitors of the canonical TGF-β signaling pathway for treatments of related diseases.

Development of a NanoBRET assay for evaluation of 14-3-3σ molecular glues

We report the development of a 384-well formatted NanoBRET assay to characterize molecular glues of 14-3-3/client interactions in living cells. The seven isoforms of 14-3-3 are dimeric hub proteins with diverse roles including transcription factor regulation and signal transduction. 14-3-3 interacts with hundreds of client proteins to regulate their function and is therefore an ideal therapeutic target when client selectivity can be achieved. We have developed the NanoBRET system for three 14-3-3σ client proteins CRAF, TAZ, and estrogen receptor α (ERα), which represent three specific binding modes. We have measured stabilization of 14-3-3σ/client complexes by molecular glues with EC50 values between 100 nM and 1 μM in cells, which align with the EC50 values calculated by fluorescence anisotropy in vitro. Developing this NanoBRET system for the hub protein 14-3-3σ allows for a streamlined approach, bypassing multiple optimization steps in the assay development process for other 14-3-3σ clients. The NanoBRET system allows for an assessment of PPI stabilization in a more physiologically relevant, cell-based environment using full-length proteins. The method is applicable to diverse protein-protein interactions (PPIs) and offers a robust platform to explore libraries of compounds for both PPI stabilizers and inhibitors.

Recent Advances in the Field of Amino Acid-Conjugated Aminoferrocenes-A Personal Perspective.

The development of turn-based inhibitors of protein-protein interactions has attracted considerable attention in medicinal chemistry. Our group has synthesized a series of peptides derived from an amino-functionalized ferrocene to investigate their potential to mimic protein turn structures. Detailed DFT and spectroscopic studies (IR, NMR, CD) have shown that, for peptides, the backbone chirality and bulkiness of the amino acid side chains determine the hydrogen-bond pattern, allowing tuning of the size of the preferred hydrogen-bonded ring in turn-folded structures. However, their biological potential is more dependent on their lipophilicity. In addition, our pioneering work on the chiroptical properties of aminoferrocene-containing peptides enables the correlation of their geometry with the sign of the CD signal in the absorption region of the ferrocene chromophore. These studies have opened up the possibility of using aminoferrocene and its derivatives as chirooptical probes for the determination of various chirality elements, such as the central chirality of amino acids and the helicity of peptide sequences.

Development of a Decafluorobiphenyl Cyclized Peptide Targeting the NEMO-IKKα/β Interaction that Enhances Cell Penetration and Attenuates Lipopolysaccharide-Induced Acute Lung Injury.

Aberrant canonical NF-κB signaling has been implicated in diseases, such as autoimmune disorders and cancer. Direct disruption of the interaction of NEMO and IKKα/β has been developed as a novel way to inhibit the overactivation of NF-κB. Peptides are a potential solution for disrupting protein-protein interactions (PPIs); however, they typically suffer from poor stability in vivo and limited tissue penetration permeability, hampering their widespread use as new chemical biology tools and potential therapeutics. In this work, decafluorobiphenyl-cysteine SNAr chemistry, molecular modeling, and biological validation allowed the development of peptide PPI inhibitors. The resulting cyclic peptide specifically inhibited canonical NF-κB signaling in vitro and in vivo, and presented positive metabolic stability, anti-inflammatory effects, and low cytotoxicity. Importantly, our results also revealed that cyclic peptides had huge potential in acute lung injury (ALI) treatment, and confirmed the role of the decafluorobiphenyl-based cyclization strategy in enhancing the biological activity of peptide NEMO-IKKα/β inhibitors. Moreover, it provided a promising method for the development of peptide-PPI inhibitors.

A Lipopeptidomimetic of Transcriptional Activation Domains Selectively Disrupts the Coactivator Med25 Protein-Protein Interactions.

Short amphipathic peptides are capable of binding to transcriptional coactivators, often targeting the same binding surfaces as native transcriptional activation domains. However, they do so with modest affinity and generally poor selectivity, limiting their utility as synthetic modulators. Here we show that incorporation of a medium-chain, branched fatty acid to the N-terminus of one such heptameric lipopeptidomimetic (LPPM-8) increases the affinity for the coactivator Med25 >20-fold (Ki >100 μM to 4 μM), rendering it an effective inhibitor of Med25 protein-protein interactions (PPIs). The lipid structure, the peptide sequence, and the C-terminal functionalization of the lipopeptidomimetic each influence the structural propensity of LPPM-8 and its effectiveness as an inhibitor. LPPM-8 engages Med25 through interaction with the H2 face of its activator interaction domain and in doing so stabilizes full-length protein in the cellular proteome. Further, genes regulated by Med25-activator PPIs are inhibited in a cell model of triple-negative breast cancer. Thus, LPPM-8 is a useful tool for studying Med25 and mediator complex biology and the results indicate that lipopeptidomimetics may be a robust source of inhibitors for activator-coactivator complexes.

Identification of small molecules affecting the interaction between human hemoglobin and Staphylococcus aureus IsdB hemophore

Human hemoglobin (Hb) is the preferred iron source of Staphylococcus aureus. This pathogenic bacterium exploits a sophisticated protein machinery called Iron-regulated surface determinant (Isd) system to bind Hb, extract and internalize heme, and finally degrade it to complete iron acquisition. IsdB, the surface exposed Hb receptor, is a proven virulence factor of S. aureus and the inhibition of its interaction with Hb can be pursued as a strategy to develop new classes of antimicrobials. To identify small molecules able to disrupt IsdB:Hb protein–protein interactions (PPIs), we carried out a structure-based virtual screening campaign and developed an ad hoc immunoassay to screen the retrieved set of commercially available compounds. Saturation-transfer difference (STD) NMR was applied to verify specific interactions of a sub-set of molecules, chosen based on their efficacy in reducing the amount of Hb bound to IsdB. Among molecules for which direct binding was verified, the best hit was submitted to ITC analysis to measure the binding affinity to Hb, which was found to be in the low micromolar range. The results demonstrate the viability of the proposed in silico/in vitro experimental pipeline to discover and test IsdB:Hb PPI inhibitors. The identified lead compound will be the starting point for future SAR and molecule optimization campaigns.

Abstract LB428: Discovery of KY-001:A Direct Inhibitor of TEAD-YAP Protein-Protein Interaction for the Treatment of Advanced Solid Tumors with Abnormal Hippo Signaling Pathway

Abstract Background:The Hippo signaling is an important pathway that plays a crucial role in the regulation of numerous biological processes. The binding of YAP/TAZ transcriptional coactivators to TEAD proteins is the core of Hippo signaling in regulating the expression of a variety of genes that mediate many cellular functions. Abnormalities of Hippo signaling including mutation deficiency, fusion and YAP/TAZ overexpression are often found in solid tumors associated with excessive proliferation, drug resistance, or tumor immune evasion. Blocking the Hippo transcription activation, particularly the TAP/TAZ and TEAD interaction, therefore is an attractive therapeutic strategy for the treatment of advanced solid tumors associated with Hippo dysregulation. Results:We have developed a small molecule inhibitor, KY-001, that can bind to TEAD protein and directly disrupt its interaction with YAP/TAZ, thereby blocking Hippo signaling pathway. KY-001 can bind to TEAD (1-4) proteins with strong YAP/TAZ competitive activities in biochemical assays and can inhibit TEAD-YAP mediated transcriptional signals in cell. KY-001 selectively and potently inhibits the proliferation of cancer cells harboring Hippo deficient mutations, such as mesothelioma, lung cancer and other cell lines. Importantly, KY-001 directly affects the expression of some target genes downstream of YAP/TAZ-TEAD transcription activation in cell. KY-001 can strongly inhibit tumor growth in a dose dependent manner against Hippo mutation mesothelioma cancers in two different xenograft mouse tumor models. In addition, KY-001 shows remarkable synergistic effects with EGFR or KRAS G12C inhibitors in mouse non-small lung cancer models, suggesting it may be a promise therapy for treating targeted therapy induced resistant tumors with Hippo dysregulation. Pre-clinical studies shows that KY-001 has good overall druggable profiles with good safety windows in several animal species. Collectively, KY-001 shows high potency and selectivity in vitro and strong anti-tumor activities against Hippo mutation deficient cancers in pre-clinical models. Conclusions:KY-001 is a small molecule drug candidate which directly inhibits the interactions between TEAD and YAP1/TAZ proteins, thereby blocking the transcriptional activation of downstream Hippo signaling. In various tumor models, KY-001 monotherapy shows good efficacy for Hippo mutation deficient tumors. KY-001 also synergizes with other targeted therapy such as EGFR or KRAS G12C inhibitors for treating non-small lung cancers. KY-001 is expected to enter clinical studies in H2, 2024 for the treatment of Hippo dysregulated solid tumors. Citation Format: Kehao Zhao, Yong Zhag, Yufang Shi, Jian Chen. Discovery of KY-001:A Direct Inhibitor of TEAD-YAP Protein-Protein Interaction for the Treatment of Advanced Solid Tumors with Abnormal Hippo Signaling Pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB428.

Discovery of Small Molecule Interleukin 17A Inhibitors with Novel Binding Mode and Stoichiometry: Optimization of DNA-Encoded Chemical Library Hits to In Vivo Active Compounds.

Dysregulation of IL17A drives numerous inflammatory and autoimmune disorders with inhibition of IL17A using antibodies proven as an effective treatment. Oral anti-IL17 therapies are an attractive alternative option, and several preclinical small molecule IL17 inhibitors have previously been described. Herein, we report the discovery of a novel class of small molecule IL17A inhibitors, identified via a DNA-encoded chemical library screen, and their subsequent optimization to provide in vivo efficacious inhibitors. These new protein-protein interaction (PPI) inhibitors bind in a previously undescribed mode in the IL17A protein with two copies binding symmetrically to the central cavities of the IL17A homodimer.

Abstract 1654: Anti-tumor activity of orally-available cyclic peptide LUNA18 through direct RAS inhibition in RAS-altered tumors

Abstract RAS is the one of the most frequently altered genes in human cancer and contributes to the growth of tumors. Recently, KRAS G12C inhibitors were approved as the first molecular targeting agents for RAS, but there are still huge unmet medical needs in RAS-mutated cancers because drugs targeting non-KRAS G12C tumors have not yet been approved and efficacy by even approved KRAS G12C inhibitors is not satisfactory. LUNA18 is the cyclic peptide that binds to RAS mutants and wildtype, including KRAS, NRAS and HRAS, thereby inhibiting protein-protein interaction (PPI) between the inactive form of RAS and GEFs (guanine nucleotide exchange factors). LUNA18 decreased the active form of KRAS and phosphorylated ERK and AKT, and cell proliferation in RAS-mutated cancer cells with nM order of cellular IC50s irrespective of tumor types and amino acid substitutions except for Q61 mutants. On the other hand, LUNA18 did not inhibit proliferation of cells with active mutations downstream of RAS (BRAF and MEK), suggesting LUNA18 selectively inhibited growth of RAS-dependent cells. Consistent with in vitro cellular activity, oral administration of LUNA18 showed durable RAS signal inhibition up to 24-48 hours and tumor regression in RAS-mutated xenograft models. In addition to KRAS-mutated cells, LUNA18 also showed in vitro and in vivo robust efficacy in KRAS-amplified cells. These data indicated LUNA18 is the versatile compound that selectively suppressed cellular signaling from RAS G12/G13 mutants as well as RAS wildtype amplification through inhibition of PPI between the inactive form of RAS and GEFs, and it can be applicable to a wide range of RAS-altered tumors. Rebound of the MAPK pathway is reported to be the one of the cause for attenuating efficacy of KRAS G12C inhibitors. We also observed the rebound of ERK activation 24-72 hours after the treatment with a KRAS G12C inhibitor in cells with the KRAS G12C mutation though suppression of KRAS activity was maintained. On the other hand, LUNA18 persistently suppressed ERK activity as well as KRAS activity up to 72 hours. The combination of the KRAS G12C inhibitor with siRNA for RAS wildtype showed durable inhibition of ERK activity, suggesting that RAS wildtype plays a key role for the rebound of ERK activity and inhibitory activity to RAS wildtype by LUNA18 contribute to the persistent inhibition of RAS signaling by LUNA18. We orally administered LUNA18 and a KRAS G12C inhibitor to a xenograft model, which showed temporal response and then acquired resistance to a KRAS G12C inhibitor, and found that LUNA18 significantly suppressed emergence of resistance to a KRAS G12C inhibitor. These results supported that the combination of the KRAS G12C inhibitor with LUNA18 could be a promising option to patients with KRAS G12C mutation by suppressing resistance to a KRAS G12C inhibitor through the suppression of rebound of the MAPK pathway. Citation Format: Hitoshi Sase, Saki Michisaka, Yukako Tachibana, Masami Hasegawa, Toshihiko Fujii, Kana Takei, Tomoko Kanei, Naoaki Murao, Shino Kuramoto, Norihito Shibahara, Atsushi Ohta, Mikimasa Tanada, Takuya Shiraishi, Hitoshi Iikura, Takehisa Kitazawa, Hiroshi Tanaka. Anti-tumor activity of orally-available cyclic peptide LUNA18 through direct RAS inhibition in RAS-altered tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1654.

PPII-AEAT: Prediction of protein-protein interaction inhibitors based on autoencoders with adversarial training

Protein-protein interactions (PPIs) have shown increasing potential as novel drug targets. The design and development of small molecule inhibitors targeting specific PPIs are crucial for the prevention and treatment of related diseases. Accordingly, effective computational methods are highly desired to meet the emerging need for the large-scale accurate prediction of PPI inhibitors. However, existing machine learning models rely heavily on the manual screening of features and lack generalizability. Here, we propose a new PPI inhibitor prediction method based on autoencoders with adversarial training (named PPII-AEAT) that can adaptively learn molecule representation to cope with different PPI targets. First, Extended-connectivity fingerprints and Mordred descriptors are employed to extract the primary features of small molecular compounds. Then, an autoencoder architecture is trained in three phases to learn high-level representations and predict inhibitory scores. We evaluate PPII-AEAT on nine PPI targets and two different tasks, including the PPI inhibitor identification task and inhibitory potency prediction task. The experimental results show that our proposed PPII-AEAT outperforms state-of-the-art methods.

Cyclic Peptide Keap1-Nrf2 Protein-Protein Interaction Inhibitors: Design, Synthesis, and In Vivo Treatment of Acute Lung Injury.

Directly blocking the Keap1-Nrf2 pathway is a promising strategy for the mitigation of acute lung injury (ALI). Peptide Keap1-Nrf2 inhibitors have been reported to have a high Keap1 binding affinity. However, these inhibitors showed weak activity in cells and/or animals. In this study, we designed a series of linear peptides from an Nrf2-based 9-mer Ac-LDEETGEFL-NH2. To improve the cellular activity, we further designed cyclic peptides based on the crystal complex of Keap1 with a linear peptide. Among them, cyclic 9-mer ZC9 targeting Keap1 showed a better affinity (KD2 = 51 nM). Specifically, it exhibited an acceptable water solubility (>38 mg/mL), better cell permeability, cell activity, and metabolic stability (serum t1/2 > 24 h). In the in vitro LPS-induced oxidative damages and ALI model, ZC9 showed significant dose-response reversal activity without apparent toxicity. In conclusion, our results suggested ZC9 as a lead cyclic peptide targeting the Keap1-Nrf2 pathway for ALI clinical treatment.

Inhibition of glycogen synthase kinase-3 enhances NRF2 protein stability, nuclear localisation and target gene transcription in pancreatic beta cells

Accumulation of reactive oxygen species (i.e., oxidative stress) is a leading cause of beta cell dysfunction and apoptosis in diabetes. NRF2 (NF-E2 p45-related factor-2) regulates the adaptation to oxidative stress, and its activity is negatively regulated by the redox-sensitive CUL3 (cullin-3) ubiquitin ligase substrate adaptor KEAP1 (Kelch-like ECH-associated protein-1). Additionally, NRF2 is repressed by the insulin-regulated Glycogen Synthase Kinase-3 (GSK3). We have demonstrated that phosphorylation of NRF2 by GSK3 enhances β-TrCP (beta-transducin repeat-containing protein) binding and ubiquitylation by CUL1 (cullin-1), resulting in increased proteasomal degradation of NRF2. Thus, we hypothesise that inhibition of GSK3 activity or β-TrCP binding upregulates NRF2 and so protects beta cells against oxidative stress. We have found that treating the pancreatic beta cell line INS-1 832/13 with the KEAP1 inhibitor TBE31 significantly enhanced NRF2 protein levels. The presence of the GSK3 inhibitor CT99021 or the β-TrCP-NRF2 protein-protein interaction inhibitor PHAR, along with TBE31, resulted in prolonged NRF2 stability and enhanced nuclear localisation (P < 0.05). TBE31-mediated induction of NRF2-target genes encoding NAD(P)H quinone oxidoreductase 1 (Nqo1), glutamate-cysteine ligase modifier (Gclm) subunit and heme oxygenase (Hmox1) was significantly enhanced by the presence of CT99021 or PHAR (P < 0.05) in both INS-1 832/13 and in isolated mouse islets. Identical results were obtained using structurally distinct GSK3 inhibitors and inhibition of KEAP1 with sulforaphane. In summary, we demonstrate that GSK3 and β-TrCP/CUL1 regulate the proteasomal degradation of NRF2, enhancing the impact of KEAP1 regulation, and so contributes to the redox status of pancreatic beta cells. Inhibition of GSK3, or β-TrCP/CUL1 binding to NRF2 may represent a strategy to protect beta cells from oxidative stress.

Abstract A002: MALT1 as a regulator of tumor progression in H3K27-altered diffuse midline glioma

Abstract A002: MALT1 as a regulator of tumor progression in H3K27-altered diffuse midline glioma

Computational Repurposing of Potential Dimerization Inhibitors against SARS-CoV-2 Main Protease

Background: The screening, design, and synthesis of various dimerization inhibitors have been an active area of interest for structure-based drug design efforts. Functionally important dimers, such as human immunodeficiency virus (HIV) protease and surviving, are being targeted for such studies over time. Computational repurposing of potential drug candidates provides a cost and time-efficient way in the drug discovery life cycle. Objective: Concerning the current coronavirus disease (COVID-19) scenario, the functionally active dimer of SARS-CoV-2 (severe acute respiratory syndrome) main protease (Mpro) is used as a target to screen possible dimerization inhibitors. Methods: A database of small molecule protein-protein interaction inhibitors was screened for the study. This study used molecular docking, followed by molecular dynamics (MD) simulation and postsimulation binding energy predictions. Results: From the selected 183 compounds, a diazene-based compound and a salicylic-type compound were identified as possible dimerization inhibitors in this study. These two compounds formed stable complexes with the Mpro during the MD simulations. The complexes formed by these two compounds were also unable to form important salt bridge interactions required for the dimerization of the protomers. Conclusion: Experimental studies on both compounds were previously conducted as dimerization inhibitors in HIV. The data led to the possibility of exploring the identified compounds as dimerization inhibitors, which could be important for SARS-CoV-2 therapeutics.

Optimization Efforts for Identification of Novel Highly Potent Keap1-Nrf2 Protein-Protein Interaction Inhibitors.

In research focused on protein-protein interaction (PPI) inhibitors, the optimization process to achieve both high inhibitory activity and favorable physicochemical properties remains challenging. Our previous study reported the discovery of novel and bioavailable Keap1-Nrf2 PPI inhibitor 8 which exhibited moderate in vivo activity in rats. In this work, we present our subsequent efforts to optimize this compound. Two distinct approaches were employed, targeting high energy water molecules and Ser602 as "hot spots" from the anchor with good aqueous solubility, metabolic stability, and membrane permeability. Through ligand efficiency (LE)-guided exploration, we identified two novel inhibitors 22 and 33 with good pharmacokinetics (PK) profiles and more potent in vivo activities, which appear to be promising chemical probes among the existing inhibitors.

Small molecule inhibitor targeting the Hsp70-Bim protein–protein interaction in estrogen receptor-positive breast cancer overcomes tamoxifen resistance

IntroductionEstrogen receptor (ER) positive patients compromise about 70% of breast cancers. Tamoxifen, an antagonist of ERα66 (the classic ER), is the most effective and the standard first-line drug. However, its efficacy is limited by the development of acquired resistance.MethodsA specific inhibitor of Hsp70-Bim protein–protein interaction (PPI), S1g-2, together with an inhibitor of Hsp70-Bag3 PPI, MKT-077 and an ATP-competitive inhibitor VER155008, were used as chemical tools. Cell viability assays, co-immunoprecipitation and gene knockdown were used to investigate the role of Hsp70 in tamoxifen resistance. A xenograft model was established in which tamoxifen-resistant breast cancer (MCF-7/TAM-R) cells maintained in the presence of 5 μM tamoxifen were subcutaneously inoculated. The anti-tumor efficiency of S1g-2 was measured after a daily injection of 0.8 mg/kg for 14 days.ResultsIt was revealed that Hsp70-Bim PPI protects ERα-positive breast cancer from tamoxifen-induced apoptosis through binding and stabilizing ERα36, rather than ERα66, resulting in sustained EGFR mRNA and protein expression. Disruption of Hsp70-Bim PPI and downregulation of ERα36 expression in tumor samples are consistent with the in vitro functions of S1g-2, resulting in about a three-fold reduction in tumor volume.ConclusionsThe in vivo activity and safety of S1g-2 illustrated that it is a potential strategy for Hsp70-Bim disruption to overcome tamoxifen-resistant ER-positive breast cancer.