Protein-protein Interaction Inhibitors Research Articles

Intersecting Peptidomics and Bioactive Peptides in Drug Therapeutics

Peptidomics is the study of total peptides that describe the functions, structures, and interactions of peptides within living organisms. It comprises bioactive peptides derived naturally or synthetically designed that exhibit various therapeutic properties against microbial infections, cancer progression, inflammation, etc. With the current state of the art, Bioinformatics tools and techniques help analyse large peptidomics data and predict peptide structure and functions. It also aids in designing peptides with enhanced stability and efficacy. Peptidomics studies are gaining importance in therapeutics as they offer increased target specificity with the least side effects. The molecular size and flexibility of peptides make them a potential drug candidate for designing protein-protein interaction inhibitors. These features increased their drug potency with the considerable increase in the number of peptide drugs available in the market for various health commodities. The present review extensively analyses the peptidomics field, focusing on different bioactive peptides and therapeutics, such as anticancer peptide drugs. Further, the review provides comprehensive information on in silico tools available for peptide research. The importance of personalised peptide medicines in disease therapy is discussed along with the case study. Further, the major limitations of peptide drugs and the different strategies to overcome those limitations are reviewed.

Discovery of WDR5-MLL1 and HDAC Dual-Target Inhibitors for the Treatment of Acute Myeloid Leukemia.

Targeting the WDR5-MLL1 protein-protein interaction (PPI) is considered to be an effective approach for the treatment of MLL-rearranged leukemia. However, interfering with WDR5-MLL1 PPI reduces methylated H3K4 levels and induces a decline in acetylated H3 levels, which may contribute to the suboptimal cellular efficacy of WDR5 inhibitors. We observed that cotreatment with WDR5-MLL1 PPI and HDAC inhibitors augmented the antiproliferative effect in MV-4-11 cells. Thus, a series of dual-target inhibitors was developed by merging the pharmacophores of the WDR5 and HDAC inhibitors. Among the developed inhibitors, compound 32d displayed an 89-fold increase in antiproliferative efficacy and induced potent cell apoptosis by impeding the DNA damage repair signaling pathway. Furthermore, the administration of 30 mg/kg of compound 32d was well tolerated, inhibiting MV-4-11 xenograft growth by 87.1%. Our investigation established the therapeutic effectiveness of the developed WDR5-MLL1/HDAC dual-target inhibitor against acute myeloid leukemia, providing a valuable tool for further exploration of crosstalk between the two targets.

Discovery of Novel Small-Molecule Inhibitors Disrupting the MTDH-SND1 Protein-Protein Interaction.

MTDH-SND1 protein-protein interaction (PPI) plays an important role in the initiation and development of tumors, and it is a target for the treatment of breast cancer. In this study, we identified and synthesized a series of novel small-molecule inhibitors of MTDH-SND1 PPI. The representative compound C19 showed potent activity against MTDH-SND1 PPI with an IC50 of 487 ± 99 nM and tight binding to the SND1-purified protein with a Kd value of 279 ± 17 nM. Compound C19 significantly degraded SND1 and downregulated downstream at the protein level. Further biological evaluations suggested that compound C19 exhibited potent activity against the proliferation of breast cancer MCF-7 cells with an IC50 value of 626 ± 27 nM, significantly inhibited invasion and migration, and induced cell apoptosis. In addition, compound C19 exhibited promising tumor growth inhibition in the xenograft model. Our study provides a potential candidate targeting MTDH-SND1 PPI for the treatment of breast cancer.

Targeting Protein-Protein Interactions in Hematologic Malignancies.

Over the last two decades, there have been extensive efforts to develop small-molecule inhibitors of protein-protein interactions (PPIs) as novel therapeutics for cancer, including hematologic malignancies. Despite the numerous challenges associated with developing PPI inhibitors, a significant number of them have advanced to clinical studies in hematologic patients in recent years. The US Food and Drug Administration approval of the very first PPI inhibitor, venetoclax, demonstrated the real clinical value of blocking protein-protein interfaces. In this review, we discuss the most successful examples of PPI inhibitors that have reached clinical studies in patients with hematologic malignancies. We also describe the challenges of blocking PPIs with small molecules, clinical resistance to such compounds, and the lessons learned from the development of successful PPI inhibitors. Overall, this review highlights the remarkable success and substantial promise of blocking PPIs in hematologic malignancies.

Computational structure-based design of antiviral peptides as potential protein-protein interaction inhibitors of rabies virus phosphoprotein and human LC8.

Rabies is a serious zoonotic disease caused by the rabies virus (RABV). Despite the successful development of vaccines and efforts made in drug discovery, rabies is incurable. Therefore, development of novel drugs is of interest to the scientific community. Antiviral peptides can be designed based on the known structures of viral proteins and their biological targets. Cytoplasmic dynein light chain LC8, one of the first identified host partners of RABV phosphoprotein (RABV P), is an essential factor for RABV transcription and replication. As part of the search for new potential drugs against rabies, we used structure-based drug design using the in silico tools. The binding site of LC8 with RABV P was used for peptide design. Four potential peptide inhibitors (Pep1-4) were selected, modeled, and docked with RABV P. The highest binding affinity was observed for the RABV P-Pep2 complex. Molecular dynamics (MD) simulations were performed and the stability of the peptides and complexes was confirmed. Finally, Pep2 can be used as a potential candidate for peptide-based antiviral therapy against RABV. The identified small peptides may prevent RABV infection based on the results of the current investigation. Further in vitro and in vivo studies are needed to confirm these results.

Structure–Activity Relationship Studies of Tetracyclic Pyrrolocarbazoles Inhibiting Heterotetrameric Protein Kinase CK2

The serine/threonine kinase CK2 (formerly known as casein kinase II) plays a crucial role in various CNS disorders and is highly expressed in various types of cancer. Therefore, inhibiting this key kinase could be promising for the treatment of these diseases. The CK2 holoenzyme is formed by the recruitment of two catalytically active CK2α and/or CK2α′ subunits by a regulatory CK2β dimer. Starting with the lead furocarbazole W16 (4) inhibiting the CK2α/CK2β interaction, analogous pyrrolocarbazoles were prepared and tested for their protein–protein interaction inhibition (PPII). The key step of the synthesis was a multicomponent Levy reaction of 2-(indolyl)acetate 6, benzaldehydes 7, and N-substituted maleimides 8. Targeted modifications were performed by the saponification of the tetracyclic ester 9a, followed by the coupling of the resulting acid 10 with diverse amines. The replacement of the O-atom of the lead furocarbazole 4 by an N-atom in pyrrolocarbazoles retained or even increased the inhibition of the CK2α/CK2β interaction. The large benzyloxazolidinyl moiety of 4 could be replaced by smaller N-substituents without the loss of the PPII. The introduction of larger substituents at the 2-position and/or at p-position of the phenyl moiety at the 10-position to increase the surface for the inhibition of the PPI did not enhance the inhibition of the CK2α/CK2β association. The strong inhibition of the CK2α/CK2β association by the histidine derivative (+)-20a (Ki = 6.1 µM) translated into a high inhibition of the kinase activity of the CK2 holoenzyme (CK2α2β2, IC50 = 2.5 µM). Thus, 20a represents a novel lead compound inhibiting CK2 via the inhibition of the association of the CK2α and Ck2β subunits.

Synthesis and biological assessment of chalcone and pyrazoline derivatives as novel inhibitor for ELF3-MED23 interaction.

HER2 overexpression significantly contributes to the aggressive nature and recurrent patterns observed in various solid tumors, notably gastric cancers. Trastuzumab, HER2-targeting monoclonal antibody drug, has shown considerable clinical success; however, readily emerging drug resistance emphasizes the pressing need for improved interventions in HER2-overexpressing cancers. To address this, we proposed targeting the protein-protein interaction (PPI) between ELF3 and MED23 as an alternative therapeutic approach to trastuzumab. In this study, we synthesized a total of 26 compounds consisting of 10 chalcones, 7 pyrazoline acetyl, and 9 pyrazoline propionyl derivatives, and evaluated their biological activity as potential ELF3-MED23 PPI inhibitors. Upon systematic analysis, candidate compound 10 was selected due to its potency in downregulating reporter gene activity of ERBB2 promoter confirmed by SEAP activity and its effect on HER2 protein and mRNA levels. Compound 10 effectively disrupted the binding interface between the ELF3 TAD domain and the 391-582 amino acid region of MED23, resulting in successful inhibition of the ELF3-MED23 PPI. This intervention led to a substantial reduction in HER2 levels and its downstream signals in the HER2-positive gastric cancer cell line. Subsequently, compound 10 induced significant apoptosis and anti-proliferative effects, demonstrating superior in vitro and in vivo anticancer activity overall. We found that the anticancer activity of compound 10 was not only restricted to trastuzumab-sensitive cases, but was also valid for trastuzumab-refractory clones. This suggests its potential as a viable therapeutic option for trastuzumab-resistant gastric cancers. In summary, compound 10 could be a novel alternative therapeutic strategy for HER2-overexpressing cancers, overcoming the limitations of trastuzumab.

Synthesis and biological assessment of chalcone and pyrazoline derivatives as novel inhibitor for ELF3-MED23 interaction

HER2 overexpression significantly contributes to the aggressive nature and recurrent patterns observed in various solid tumors, notably gastric cancers. Trastuzumab, HER2-targeting monoclonal antibody drug, has shown considerable clinical success; however, readily emerging drug resistance emphasizes the pressing need for improved interventions in HER2-overexpressing cancers. To address this, we proposed targeting the protein-protein interaction (PPI) between ELF3 and MED23 as an alternative therapeutic approach to trastuzumab. In this study, we synthesized a total of 26 compounds consisting of 10 chalcones, 7 pyrazoline acetyl, and 9 pyrazoline propionyl derivatives, and evaluated their biological activity as potential ELF3-MED23 PPI inhibitors. Upon systematic analysis, candidate compound 10 was selected due to its potency in downregulating reporter gene activity of ERBB2 promoter confirmed by SEAP activity and its effect on HER2 protein and mRNA levels. Compound 10 effectively disrupted the binding interface between the ELF3 TAD domain and the 391–582 amino acid region of MED23, resulting in successful inhibition of the ELF3-MED23 PPI. This intervention led to a substantial reduction in HER2 levels and its downstream signals in the HER2-positive gastric cancer cell line. Subsequently, compound 10 induced significant apoptosis and anti-proliferative effects, demonstrating superior in vitro and in vivo anticancer activity overall. We found that the anticancer activity of compound 10 was not only restricted to trastuzumab-sensitive cases, but was also valid for trastuzumab-refractory clones. This suggests its potential as a viable therapeutic option for trastuzumab-resistant gastric cancers. In summary, compound 10 could be a novel alternative therapeutic strategy for HER2-overexpressing cancers, overcoming the limitations of trastuzumab.

Advances in Protein–Protein Interaction Inhibitors (PPIs): Drug Discovery Strategies

Advances in Protein–Protein Interaction Inhibitors (PPIs): Drug Discovery Strategies

Discovery of Propionic Acid Derivatives with a 5-THIQ Core as Potent and Orally Bioavailable Keap1-Nrf2 Protein-Protein Interaction Inhibitors for Acute Kidney Injury.

Keap1 plays a crucial role in regulating the Nrf2-mediated cytoprotective response and is increasingly targeted for oxidative stress-related diseases. Using small molecules to disrupt the Keap1-Nrf2 protein-protein interaction (PPI) has emerged as a new strategy for developing Nrf2 activators. Through extensive structure-activity relationship studies, we identified compound 56, which features a unique 5-tetrahydroisoquinoline scaffold and acts as a potent inhibitor of the Keap1-Nrf2 PPI. Compound 56 exhibited significant inhibitory activity (IC50 = 16.0 nM) and tight Keap1 binding affinity (Kd = 3.07 nM), along with acceptable oral bioavailability (F = 20%). Notably, 56 enhanced antioxidant defenses in HK-2 renal tubular epithelial cells and significantly reduced plasma creatinine and blood urea nitrogen levels in acute kidney injury (AKI) mice. These findings collectively position compound 56 as a promising candidate for the treatment of AKI.

Förster Resonance Energy Transfer Measurements in Living Bacteria for Interaction Studies of BamA with BamD and Inhibitor Identification.

The β-barrel assembly machinery (BAM) is a multimeric protein complex responsible for the folding of outer membrane proteins in gram-negative bacteria. It is essential for cell survival and outer membrane integrity. Therefore, it is of impact in the context of antibiotic resistance and can serve as a target for the development of new antibiotics. The interaction between two of its subunits, BamA and BamD, is essential for its function. Here, a FRET-based assay to quantify the affinity between these two proteins in living bacterial cells is presented. The method was applied to identify two interaction hotspots at the binding interface. BamDY184 was identified to significantly contribute to the binding between both proteins through hydrophobic interactions and hydrogen bonding. Additionally, two salt bridges formed between BamDR94, BamDR97, and BamAE127 contributed substantially to the binding of BamA to BamD as well. Two peptides (RFIRLN and VAEYYTER) that mimic the amino acid sequence of BamD around the identified hotspots were shown to inhibit the interaction between BamA and BamD in a dose-dependent manner in the upper micromolar range. These two peptides can potentially act as antibiotic enhancers. This shows that the BamA-BamD interaction site can be addressed for the design of protein-protein interaction inhibitors. Additionally, the method, as presented in this study, can be used for further functional studies on interactions within the BAM complex.

Modulators of the Hop-HSP90 Protein-Protein Interaction Disrupt KSHV Lytic Replication.

The central role of the chaperome in maintaining cellular proteostasis has seen numerous viral families evolve to parasitically exploit host chaperones in their life cycle. The HSP90 chaperone protein and its cochaperone Hop have both individually been shown to be essential factors for Kaposi sarcoma-associated herpesvirus (KSHV) lytic replication. Given the fundamental regulatory role that protein-protein interactions (PPIs) play in cellular biology, we reasoned that disrupting the Hop-HSP90 PPI may provide a new host-based target for inhibiting KSHV lytic replication. This study expands upon a previous report of non-natural peptides, which were found to disrupt the association between the HopTPR2A domain and its interacting HSP90CTD. Here, in addition to providing insight into the structure-activity relationships of PPI inhibition, we show disruption of the full-length Hop-HSP90 PPI. The inhibitory peptides selectively engaged the HopTPR2A domain in cell lysates and when tethered to a cell-penetrating peptide acted as noncytotoxic inhibitors of KSHV lytic replication by lowering the viral load, preventing the production of infectious virions, and reducing the expression of KSHV lytic genes. In addition to tentative evidence of Hop-HSP90 PPI as a much-needed target for KSHV drug discovery, this study represents an important step in understanding viral interactions with the host proteostasis machinery.

Peptide-Based Inhibitors of Protein-Protein Interactions (PPIs): A Case Study on the Interaction Between SARS-CoV-2 Spike Protein and Human Angiotensin-Converting Enzyme 2 (hACE2).

Protein-protein interactions (PPIs) are fundamental to many critical biological processes and are crucial in mediating essential cellular functions across diverse organisms, including bacteria, parasites, and viruses. A notable example is the interaction between the SARS-CoV-2 spike (S) protein and the human angiotensin-converting enzyme 2 (hACE2), which initiates a series of events leading to viral replication. Interrupting this interaction offers a promising strategy for blocking or significantly reducing infection, highlighting its potential as a target for anti-SARS-CoV-2 therapies. This review focuses on the hACE2 and SARS-CoV-2 spike protein interaction, exemplifying the latest advancements in peptide-based strategies for developing PPI inhibitors. We discuss various approaches for creating peptide-based inhibitors that target this critical interaction, aiming to provide potential treatments for COVID-19.

Inhibition of Lysosomal Cathepsin A and Neuraminidase 1 Interaction by Anti-Obesity Cyclic Peptide.

Chronic inflammation in adipose tissue is associated with metabolic disorders such as obesity and type 2 diabetes. Novel small molecules targeting adipocyte differentiation and fat accumulation offer potential for new anti-inflammatory and anti-obesity drugs. Here we show that the marine cyclic heptapeptide stylissatin A and its analogs (SAs) inhibit membranous neuraminidase 1 (Neu1) function by interacting with lysosomal protective protein cathepsin A (PPCA). Neu1 has been less explored as a therapeutic target due to the genetic defects leading to neurodegenerative disorders. However, unlike traditional neuraminidase inhibitors, SAs don't directly bind to Neu1 but modulate the molecular chaperone activity of PPCA. SAs caused degradation of perilipin 1 around lipid droplets and inhibited fat accumulation, along with decrease in membranous Neu1. Molecular docking and molecular dynamics simulations revealed that SAs interacted with activated PPCA at the Neu1 binding site. Focusing on this newfound protein-protein interaction inhibition mechanism could lead to the development of pharmaceuticals with fewer side effects.

Probing hot spots of protein-protein interactions mediated by the safety-belt region of REV7

REV7 is a HORMA (Hop1, Rev7, Mad2) family adaptor protein best known as an accessory subunit of the translesion synthesis (TLS) DNA polymerase ζ (Polζ). In this role, REV7 binds REV3, the catalytic subunit of Polζ, by locking REV7-binding motifs (RBMs) in REV3 underneath the REV7 safety-belt loop. The same mechanism is used by REV7 to interact with RBMs from other proteins in DNA damage response (DDR) and mitosis. Because of the importance of REV7 for TLS and other DDR pathways, targeting REV7:RBM protein-protein interactions (PPIs) with small molecules has emerged as a strategy to enhance cancer response to genotoxic chemotherapy. To identify druggable pockets at the REV7:RBM interface, we performed computational analyses of REV7 complexed with several RBM partners. The contributions of different interface regions to REV7:RBM stabilization were corroborated experimentally. These studies provide insights into key intermolecular interactions and establish targetable regions of REV7 for the design of REV7:RBM PPI inhibitors.

Targeting HSP90 for Cancer Therapy: Current Progress and Emerging Prospects.

Heat shock protein 90 (HSP90), a highly conserved member of the heat shock protein family, regulates various proteins and signaling pathways involved in cancer, making it a promising target for cancer therapy. Traditional HSP90 inhibitors have demonstrated significant antitumor potential in preclinical trials, with over 20 compounds advancing to clinical trials and showing promising results. However, the limited clinical efficacy and shared toxicity of these inhibitors restrict their further clinical use. Encouragingly, developing novel inhibitors using conventional medicinal chemistry approaches─such as selective inhibitors, dual inhibitors, protein-protein interaction inhibitors, and proteolysis-targeting chimeras─is expected to address these challenges. Notably, the selective inhibitor TAS-116 has already been successfully marketed. In this Perspective, we summarize the structure, biological functions, and roles of HSP90 in cancer, analyze the clinical status of HSP90 inhibitors, and highlight the latest advancements in novel strategies, offering insights into their future development.

Selective Aurora A-TPX2 Interaction Inhibitors Have In Vivo Efficacy as Targeted Antimitotic Agents.

Aurora A kinase, a cell division regulator, is frequently overexpressed in various cancers, provoking genome instability and resistance to antimitotic chemotherapy. Localization and enzymatic activity of Aurora A are regulated by its interaction with the spindle assembly factor TPX2. We have used fragment-based, structure-guided lead discovery to develop small molecule inhibitors of the Aurora A-TPX2 protein-protein interaction (PPI). Our lead compound, CAM2602, inhibits Aurora A:TPX2 interaction, binding Aurora A with 19 nM affinity. CAM2602 exhibits oral bioavailability, causes pharmacodynamic biomarker modulation, and arrests the growth of tumor xenografts. CAM2602 acts by a novel mechanism compared to ATP-competitive inhibitors and is highly specific to Aurora A over Aurora B. Consistent with our finding that Aurora A overexpression drives taxane resistance, these inhibitors synergize with paclitaxel to suppress the outgrowth of pancreatic cancer cells. Our results provide a blueprint for targeting the Aurora A-TPX2 PPI for cancer therapy and suggest a promising clinical utility for this mode of action.

Targeting Grb2 SH3 Domains with Affimer Proteins Provides Novel Insights into Ras Signalling Modulation.

Src homology 3 (SH3) domains play a critical role in mediating protein-protein interactions (PPIs) involved in cell proliferation, migration, and the cytoskeleton. Despite their abundance in the human proteome, the functions and molecular interactions of many SH3 domains remain unknown, and this is in part due to the lack of SH3-domain-specific reagents available for their study. Affimer proteins have been developed as affinity reagents targeting a diverse range of targets, including those involved in PPIs. In this study, Affimer proteins were isolated against both the N- and C-terminal SH3 domains (NSH3 and CSH3) of growth-factor-receptor-bound protein 2 (Grb2), an adapter protein that provides a critical link between cell surface receptors and Ras signalling pathways. Targeting the CSH3 alone for the inhibition of PPIs appeared sufficient for curtailing Ras signalling in mammalian cell lines stimulated with human epidermal growth factor (EGF), which conflicts with the notion that the predominant interactions with Ras activating Son of sevenless (SOS) occur via the NSH3 domain. This result supports a model in which allosteric mechanisms involved in Grb2-SOS1 interaction modulate Ras activation.

Computational Assessment of Carotenoids as Keap1-Nrf2 Protein-Protein Interaction Inhibitors: Implications for Antioxidant Strategies.

The Keap1-Nrf2 pathway is an essential system that maintains redox homeostasis and modulates key metabolic processes, including metabolism of amino acids to promote the synthesis of antioxidant enzymes. Inhibitors of the protein-protein interaction (PPI) between Keap1 and Nrf2 have emerged as a promising strategy for developing novel classes of antioxidant agents that selectively activate this pathway without off-target effects. Carotenoids, a large family of lipophilic isoprenoids synthesized by all photosynthetic organisms, are well-known for their antioxidant activities. However, the ability of carotenoids to inhibit the Keap1-Nrf2 PPI through the involvement of specific amino acid residues has not yet been revealed. We utilized molecular docking, molecular dynamic simulations, and pharmacokinetic prediction techniques to investigate the potential of eight oxygenated carotenoids, known as xanthophylls, to inhibit Keap1. Among the compounds investigated, fucoxanthin and astaxanthin established multiple hydrogen-bonding and hydrophobic interactions within the Kelch domain of Keap1, showing remarkable binding affinities. Furthermore, fucoxanthin and astaxanthin displayed the ability toform astable complex with Keap1 and fit into the binding pocket of its Kelch domain. These analyses led to the identification of critical amino acid residues in the binding pocket of Keap1 which are involved in the interaction with carotenoid xanthophylls. Our analyses further revealed that fucoxanthin and astaxanthin demonstrate a favorable safety profile and possess pharmacokinetic properties consistent with acceptable drug-like characteristics. These findings lay the preliminary foundation for developing a novel class of Keap1-Nrf2 PPI inhibitors with potential applications against oxidative stress-related diseases.

Exploring a New Generation of Pyrimidine and Pyridine Derivatives as Anti-Influenza Agents Targeting the Polymerase PA-PB1 Subunits Interaction.

The limited range of available flu treatments due to virus mutations and drug resistance have prompted the search for new therapies. RNA-dependent RNA polymerase (RdRp) is a heterotrimeric complex of three subunits, i.e., polymerase acidic protein (PA) and polymerase basic proteins 1 and 2 (PB1 and PB2). It is widely recognized as one of the most promising anti-flu targets because of its critical role in influenza infection and high amino acid conservation. In particular, the disruption of RdRp complex assembly through protein-protein interaction (PPI) inhibition has emerged as a valuable strategy for discovering a new therapy. Our group previously identified the 3-cyano-4,6-diphenyl-pyridine core as a privileged scaffold for developing PA-PB1 PPI inhibitors. Encouraged by these findings, we synthesized a small library of pyridine and pyrimidine derivatives decorated with a thio-N-(m-tolyl)acetamide side chain (compounds 2a-n) or several amino acid groups (compounds 3a-n) at the C2 position. Interestingly, derivative 2d, characterized by a pyrimidine core and a phenyl and 4-chloro phenyl ring at the C4 and C6 positions, respectively, showed an IC50 value of 90.1 μM in PA-PB1 ELISA, an EC50 value of 2.8 μM in PRA, and a favorable cytotoxic profile, emerging as a significant breakthrough in the pursuit of new PPI inhibitors. A molecular modeling study was also completed as part of this project, allowing us to clarify the biological profile of these compounds.