Protein-protein Interaction Inhibitors Research Articles

Specific inhibition of an anticancer target, polo-like kinase 1, by allosterically dismantling its mechanism of substrate recognition

Polo-like kinase 1 (Plk1) is considered an attractive target for anticancer therapy. Over the years, studies on the noncatalytic polo-box domain (PBD) of Plk1 have raised the expectation of generating highly specific protein-protein interaction inhibitors. However, the molecular nature of the canonical PBD-dependent interaction, which requires extensive water network-mediated interactions with its phospholigands, has hampered efforts to identify small molecules suitable for Plk1 PBD drug discovery. Here, we report the identification of the first allosteric inhibitor of Plk1 PBD, called Allopole, a prodrug that can disrupt intracellular interactions between PBD and its cognate phospholigands, delocalize Plk1 from centrosomes and kinetochores, and induce mitotic block and cancer cell killing. At the structural level, its unmasked active form, Allopole-A, bound to a deep Trp-Phe-lined pocket occluded by a latch-like loop, whose adjoining region was required for securely retaining a ligand anchored to the phospho-binding cleft. Allopole-A binding completely dislodged the L2 loop, an event that appeared sufficient to trigger the dissociation of a phospholigand and inhibit PBD-dependent Plk1 function during mitosis. Given Allopole's high specificity and antiproliferative potency, this study is expected to open an unexplored avenue for developing Plk1 PBD-specific anticancer therapeutic agents.

Molecular Design of Cyclic Peptides with Cell Membrane Permeability and Development of MDMX-p53 Inhibitor.

Cyclic peptides have been expected to be one of the modalities of intracellular protein-protein interaction (PPI) inhibitors, but they are generally known to have low cell membrane permeability. In this study, we focused on the conformation of cyclic peptides in the cell membrane to determine the requirement for their cell membrane permeability through passive diffusion. Utilizing the requirement, we searched for structures with high affinity for MDMX via computational chemistry and acquired cyclic peptide 19 (Papp = 0.80 × 10-6 cm s-1, IC50 = 0.07 μM).

State of the art in protein-protein interactions within the fungi kingdom.

Proteins rarely exert their function by themselves. Protein-protein interactions (PPIs) regulate virtually every biological process that takes place in a cell. Such interactions are targets for new therapeutic agents against all sorts of diseases, through the screening and design of a variety of inhibitors. Here we discuss several aspects of PPIs that contribute to prediction of protein function and drug discovery. As the high-throughput techniques continue to release biological data, targets for fungal therapeutics that rely on PPIs are being proposed worldwide. Computational approaches have reduced the time taken to develop new therapeutic approaches. The near future brings the possibility of developing new PPI and interaction network inhibitors and a revolution in the way we treat fungal diseases.

Identification of α-Tocopherol succinate as an RFFL-substrate interaction inhibitor inducing peripheral CFTR stabilization and apoptosis

The E3 ubiquitin ligase RFFL is an apoptotic inhibitor highly expressed in cancers and its knockdown suppresses cancer cell growth and sensitizes to chemotherapy. RFFL also participates in peripheral protein quality control which removes the functional cell surface ΔF508-CFTR channel and reduces the efficacy of pharmaceutical therapy for cystic fibrosis (CF). Although RFFL inhibitors have therapeutic potential for both cancer and CF, they remain undiscovered. Here, a chemical array screening has identified α-tocopherol succinate (αTOS) as an RFFL ligand. NMR analysis revealed that αTOS directly binds to RFFL’s substrate-binding region without affecting the E3 enzymatic activity. Consequently, αTOS inhibits the RFFL-substrate interaction, ΔF508-CFTR ubiquitination and elimination from the plasma membrane of epithelial cells, resulting in the increased functional CFTR channel. Among the α-tocopherol (αTOL) analogs we tested, only αTOS inhibited the RFFL-substrate interaction and increased the cell surface ΔF508-CFTR, depending on RFFL expression. Similarly, the unique proapoptotic effect of αTOS was dependent on RFFL expression. Thus, unlike other αTOL analogs, αTOS acts as an RFFL protein-protein interaction inhibitor which may explain its unique biological properties among αTOL analogs. Moreover, αTOS may act as a CFTR stabilizer, a novel class of drugs that extend cell surface ΔF508-CFTR lifetime.

Effect of Colchicine Binding Site Inhibitors on the Tubulin Intersubunit Interaction.

Microtubules are dynamic, non-covalent polymers consisting of α- and β-tubulin subunits that are involved in a wide range of intracellular processes. The polymerization and dynamics of microtubules are regulated by many factors, including small molecules that interact with different sites on the tubulin dimer. Colchicine binding site inhibitors (CBSIs) destabilize microtubules and inhibit tubulin polymerization, leading to cell cycle arrest. Because of their therapeutic potential, the molecular mechanism of CBSI function is an area of active research. Nevertheless, important details of this mechanism have yet to be resolved. In this study, we use atomistic molecular dynamics simulations to show that the binding of CBSIs to the tubulin heterodimer leads to the weakening of tubulin intersubunit interaction. Using atomistic molecular dynamics simulations and binding free energy calculations, we show that CBSIs act as protein-protein interaction inhibitors and destabilize interlinkage between α and β subunits, which is crucial for longitudinal contacts in the microtubule lattice. Our results offer new insight into the mechanisms of microtubule polymerization inhibition by colchicine and its analogs.

Potentiating Tweezer Affinity to a Protein Interface with Sequence-Defined Macromolecules on Nanoparticles.

Survivin, a well-known member of the inhibitor of apoptosis protein family, is upregulated in many cancer cells, which is associated with resistance to chemotherapy. To circumvent this, inhibitors are currently being developed to interfere with the nuclear export of survivin by targeting its protein-protein interaction (PPI) with the export receptor CRM1. Here, we combine for the first time a supramolecular tweezer motif, sequence-defined macromolecular scaffolds, and ultrasmall Au nanoparticles (us-AuNPs) to tailor a high avidity inhibitor targeting the survivin-CRM1 interaction. A series of biophysical and biochemical experiments, including surface plasmon resonance measurements and their multivalent evaluation by EVILFIT, reveal that for divalent macromolecular constructs with increasing linker distance, the longest linkers show superior affinity, slower dissociation, as well as more efficient PPI inhibition. As a drawback, these macromolecular tweezer conjugates do not enter cells, a critical feature for potential applications. The problem is solved by immobilizing the tweezer conjugates onto us-AuNPs, which enables efficient transport into HeLa cells. On the nanoparticles, the tweezer valency rises from 2 to 16 and produces a 100-fold avidity increase. The hierarchical combination of different scaffolds and controlled multivalent presentation of supramolecular binders was the key to the development of highly efficient survivin-CRM1 competitors. This concept may also be useful for other PPIs.

Generating Potential Protein-Protein Interaction Inhibitor Molecules Based on Physicochemical Properties.

Protein-protein interactions (PPIs) are associated with various diseases; hence, they are important targets in drug discovery. However, the physicochemical empirical properties of PPI-targeted drugs are distinct from those of conventional small molecule oral pharmaceuticals, which adhere to the "rule of five (RO5)". Therefore, developing PPI-targeted drugs using conventional methods, such as molecular generation models, is challenging. In this study, we propose a molecular generation model based on deep reinforcement learning that is specialized for the production of PPI inhibitors. By introducing a scoring function that can represent the properties of PPI inhibitors, we successfully generated potential PPI inhibitor compounds. These newly constructed virtual compounds possess the desired properties for PPI inhibitors, and they show similarity to commercially available PPI libraries. The virtual compounds are freely available as a virtual library.

Sea Sail Venom: a Blessing in Disguise

This opinion article reports a pioneering effort in its field, indicating the potential of conotoxins as pharmacological agents for anti-adhesion adjunct therapy in the context of malaria. In a similar vein, the potential application of conotoxins as inhibitors of protein-protein interactions holds promise for the mitigation of emerging diseases such as AIDS and COVID- 19.

Medicinal Chemistry Insights into the Development of Small-Molecule Kelch-Like ECH-Associated Protein 1-Nuclear Factor Erythroid 2-Related Factor 2 (Keap1-Nrf2) Protein-Protein Interaction Inhibitors.

Oxidative stress has been implicated in a wide range of pathological conditions. The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) exerts a central role in regulating the cellular defense system against oxidative and electrophilic insults. Nonelectrophilic inhibition of the protein-protein interaction (PPI) between Kelch-like ECH-associated protein 1 (Keap1) and Nrf2 has become a promising approach to activate Nrf2. Recently, multiple drug discovery strategies have facilitated the development of small-molecule Keap1-Nrf2 PPI inhibitors with potent activity and favorable drug-like properties. In this Perspective, we summarize the latest progress of small-molecule Keap1-Nrf2 PPI inhibitors from medicinal chemistry insights and discuss future prospects and challenges in this field.

Peptide-Hypervalent Iodine Reagent Chimeras: Enabling Peptide Functionalization and Macrocyclization.

Herein, we report a novel strategy for the modification of peptides based on the introduction of highly reactive hypervalent iodine reagents-ethynylbenziodoxolones (EBXs)-onto peptides. These peptide-EBXs can be readily accessed, by both solution- and solid-phase peptide synthesis (SPPS). They can be used to couple the peptide to other peptides or a protein through reaction with Cys, leading to thioalkynes in organic solvents and hypervalent iodine adducts in water buffer. Furthermore, a photocatalytic decarboxylative coupling to the C-terminus of peptides was developed using an organic dye and was also successful in an intramolecular fashion, leading to macrocyclic peptides with unprecedented crosslinking. A rigid linear aryl alkyne linker was essential to achieve high affinity for Keap1 at the Nrf2 binding site with potential protein-protein interaction inhibition.

Multivalent Molecular Tweezers Disrupt the Essential NDC80 Interaction with Microtubules.

Binding of microtubule filaments by the conserved Ndc80 protein is required for kinetochore-microtubule attachments in cells and the successful distribution of the genetic material during cell division. The reversible inhibition of microtubule binding is an important aspect of the physiological error correction process. Small molecule inhibitors of protein-protein interactions involving Ndc80 are therefore highly desirable, both for mechanistic studies of chromosome segregation and also for their potential therapeutic value. Here, we report on a novel strategy to develop rationally designed inhibitors of the Ndc80 Calponin-homology domain using Supramolecular Chemistry. With a multiple-click approach, lysine-specific molecular tweezers were assembled to form covalently fused dimers to pentamers with a different overall size and preorganization/stiffness. We identified two dimers and a trimer as efficient Ndc80 CH-domain binders and have shown that they disrupt the interaction between Ndc80 and microtubules at low micromolar concentrations without affecting microtubule dynamics. NMR spectroscopy allowed us to identify the biologically important lysine residues 160 and 204 as preferred tweezer interaction sites. Enhanced sampling molecular dynamics simulations provided a rationale for the binding mode of multivalent tweezers and the role of pre-organization and secondary interactions in targeting multiple lysine residues across a protein surface.

Nrf2 as a Potential Therapeutic Target for Traumatic Brain Injury.

In this review, we discuss the possibility and feasibility of nuclear factor erythroid 2-related factor 2 (Nrf2) as a therapeutic target to minimize the devastating effects of a brain injury. To complete this review, comprehensive literature searches were conducted in MEDLINE, PubMed, Embase, and PsycINFO databases for English scientific peer-reviewed articles through December 2022. This short review addressed the different sources of oxidative stress and its effects on blood-brain barrier (BBB) dysfunction, mitochondrial damage, and changes in a variety of inflammatory molecules associated with central nervous system (CNS) injury. At last, we explained the potential efficacy of the Nrf2 transcription factor in reducing oxidative stress-mediated secondary damages after a CNS injury. The role of CPUY192018, an inhibitor of Nrf2-Keap1 protein-protein interaction in protecting the injured brain cells is given as evidence of Nrf2's role in activating antioxidant genes. Overall, the scope of Nrf2 in developing therapeutic interventions for a variety of pathophysiological conditions associated with CNS injury-induced free radical/inflammatory signaling is acknowledged. Nrf2 has a widespread application in basic and clinical neuroscience for understanding and treating free radical/inflammatory signaling disorders, including neurological diseases. The development of innovative therapeutic strategies using Nrf2-inducing agents can be applied to reduce the complications of TBI before advancing it to posttraumatic stress disorder (PTSD).

Development of novel PEX5-PEX14 protein-protein interaction (PPI) inhibitors based on an oxopiperazine template

Protein-protein interactions (PPIs) constitute an important but challenging class of molecular targets for small molecules. The PEX5-PEX14 PPI has been shown to play a critical role in glycosome biogenesis and its disruption impairs the metabolism in Trpanosoma parasites, eventually leading to their death. Therefore, this PPI is a potential molecular target for new drugs against diseases caused by Trypanosoma infections. Here, we report a new class of peptidomimetic scaffolds to target the PEX5-PEX14 PPI. The molecular design was based on an oxopiperazine template for the α-helical mimetics. A structural simplification along with modifications of the central oxopiperazine scaffold and addressing the lipophilic interactions led to the development of peptidomimetics that inhibit PEX5-TbPEX14 PPI and display cellular activity against T. b. brucei. This approach provides an alternative approach towards the development of trypanocidal agents and may be generally useful for the design of helical mimetics as PPI inhibitors.

A potent phosphodiester Keap1-Nrf2 protein-protein interaction inhibitor as the efficient treatment of Alzheimer's disease

The Keap1-Nrf2 pathway has been established as a therapeutic target for Alzheimer's disease (AD). Directly inhibiting the protein-protein interaction (PPI) between Keap1 and Nrf2 has been reported as an effective strategy for treating AD. Our group has validated this in an AD mouse model for the first time using the inhibitor 1,4-diaminonaphthalene NXPZ-2 with high concentrations. In the present study, we reported a new phosphodiester containing diaminonaphthalene compound, POZL, designed to target the PPI interface using a structure-based design strategy to combat oxidative stress in AD pathogenesis. Our crystallographic verification confirms that POZL shows potent Keap1-Nrf2 inhibition. Remarkably, POZL showed its high in vivo anti-AD efficacy at a much lower dosage compared to NXPZ-2 in the transgenic APP/PS1 AD mouse model. POZL treatment in the transgenic mice could effectively ameliorate learning and memory dysfunction by promoting the Nrf2 nuclear translocation. As a result, the oxidative stress and AD biomarker expression such as BACE1 and hyperphosphorylation of Tau were significantly reduced, and the synaptic function was recovered. HE and Nissl staining confirmed that POZL improved brain tissue pathological changes by enhancing neuron quantity and function. Furthermore, it was confirmed that POZL could effectively reverse Aβ-caused synaptic damage by activating Nrf2 in primary cultured cortical neurons. Collectively, our findings demonstrated that the phosphodiester diaminonaphthalene Keap1-Nrf2 PPI inhibitor could be regarded as a promising preclinical candidate of AD.

Peptide-mediated inhibition of the transcriptional regulator Elongin BC induces apoptosis in cancer cells

Inhibition of protein-protein interactions (PPIs) via designed peptides is an effective strategy to perturb their biological functions. The Elongin BC heterodimer (ELOB/C) binds to a BC-box motif and is essential for cancer cell growth. Here, we report a peptide that mimics the high-affinity BC-box of the PRC2-associated protein EPOP. This peptide tightly binds to the ELOB/C dimer (kD= 0.46± 0.02nM) and blocks the association of ELOB/C with its interaction partners, both invitro and in the cellular environment. Cancer cells treated with our peptide inhibitor showed decreased cell viability, increased apoptosis, and perturbed gene expression. Therefore, our work proposes that blocking the BC-box-binding pocket of ELOB/C is a feasible strategy to impair its function and inhibit cancer cell growth. Our peptide inhibitor promises novel mechanistic insights into the biological function of the ELOB/C dimer and offers a starting point for therapeutics linked to ELOB/C dysfunction.

Novel Medicinal Chemistry Strategies Targeting CDK5 for Drug Discovery.

Cyclin-dependent kinase 5 (CDK5) protein plays an important role not only in the central nervous system but also in the periphery, including immune response, regulation of insulin secretion, and cancer development and progression. Consequently, targeting the CDK5 protein is a potential strategy for the treatment of many diseases, especially cancer and neurodegenerative diseases. To date, numerous pan-CDK inhibitors have entered clinical trials. Nevertheless, limited clinical efficacy and severe adverse effects have prompted the application of new techniques to optimize clinical efficacy and minimize adverse events. In this Perspective, we highlight the protein properties, biofunctions, relevant signaling pathways, and associations with cancer development and proliferation of CDK5, and analyze the clinical status of pan-CDK inhibitors and the preclinical status of CDK5-specific inhibitors. In addition, CDK5-selective inhibitors, protein-protein interaction inhibitors, proteolytic-targeting chimera (PROTAC) degraders, and dual-target CDK5 inhibitors are discussed.

Hsp70-Bim interaction facilitates mitophagy by recruiting parkin and TOMM20 into a complex

BackgroundFor cancer therapy, the identification of both selective autophagy targets and small molecules that specifically regulate autophagy is greatly needed. Heat shock protein 70 (Hsp70) is a recently discovered BH3 receptor that forms a protein‒protein interaction (PPI) with Bcl-2-interacting mediator of cell death (Bim). Herein, a specific inhibitor of the Hsp70-Bim PPI, S1g-2, and its analog S1, which is a Bcl-2-Bim disruptor, were used as chemical tools to explore the role of Hsp70-Bim PPI in regulating mitophagy.MethodsCo-immunoprecipitation and immunofluorescence assays were used to determine protein interactions and colocalization patterns. Organelle purification and immunodetection of LC3-II/LC3-I on mitochondria, endoplasmic reticulum (ER) and Golgi were applied to identify specific types of autophagy. Cell-based and in vitro ubiquitination studies were used to study the role of the Hsp70-Bim PPI in parkin-mediated ubiquitination of outer mitochondrial membrane 20 (TOMM20).ResultsWe found that after the establishment of their PPI, Hsp70 and Bim form a complex with parkin and TOMM20, which in turn facilitates parkin translocation to mitochondria, TOMM20 ubiquitination and mitophagic flux independent of Bax/Bak. Moreover, S1g-2 selectively inhibits stress-induced mitophagy without interfering with basal autophagy.ConclusionsThe findings highlight the dual protective function of the Hsp70-Bim PPI in regulating both mitophagy and apoptosis. S1g-2 is thus a newly discovered antitumor drug candidate that drives both mitophagy and cell death via apoptosis.Graphical

SVSBI: sequence-based virtual screening of biomolecular interactions

Virtual screening (VS) is a critical technique in understanding biomolecular interactions, particularly in drug design and discovery. However, the accuracy of current VS models heavily relies on three-dimensional (3D) structures obtained through molecular docking, which is often unreliable due to the low accuracy. To address this issue, we introduce a sequence-based virtual screening (SVS) as another generation of VS models that utilize advanced natural language processing (NLP) algorithms and optimized deep K-embedding strategies to encode biomolecular interactions without relying on 3D structure-based docking. We demonstrate that SVS outperforms state-of-the-art performance for four regression datasets involving protein-ligand binding, protein-protein, protein-nucleic acid binding, and ligand inhibition of protein-protein interactions and five classification datasets for protein-protein interactions in five biological species. SVS has the potential to transform current practices in drug discovery and protein engineering.

Design, synthesis, and biological evaluation of furan-bearing pyrazolo[3,4-b]pyridines as novel inhibitors of CDK2 and P53-MDM2 protein-protein interaction.

The novel series of furan-bearing pyrazolo[3,4-b]pyridines were designed as cyclin-dependent kinase 2 (CDK2) inhibitors and as p53-murine double minute 2(MDM2) inhibitors. The newly synthesized compounds were screened for their antiproliferative activity towardhepatocellular carcinoma (HepG2) and breast cancer (MCF7) cell lines. The most active compounds on both cell lines were additionally evaluated for their in vitro CDK2inhibitory activity. Compounds 7b and 12f displayed enhanced activity (half-maximal inhibitory concentration[IC50 ] = 0.46 and 0.27 µM, respectively) in comparison to the standard roscovitine (IC50 = 1.41 ± 0.03 µM), in addition to, cell cycle arrest at S phase and G1/S transition phase in MCF7 cells treated with both compounds, respectively. Moreover, the most active spiro-oxindole derivative against MCF7 cell line, 16a, exhibited enhanced inhibitory activity against p53-MDM2 interaction in vitro (IC50 = 3.09 ± 0.12 µM) compared to nutlin, and increased the levels of both p53 and p21 by nearly fourfold in comparison to the negative control. Molecular docking studies demonstrated the plausible interaction patterns of the most potent derivatives 17b and 12f in the CDK2 binding pocket and the spiro-oxindole 16a with p53-MDM2 complex, respectively. Consequently, the new chemotypes 7b, 12f, and 16a can be presented as promising antitumor hits for further studies and optimization.

Discovery of AI-2 Quorum Sensing Inhibitors Targeting the LsrK/HPr Protein-Protein Interaction Site by Molecular Dynamics Simulation, Virtual Screening, and Bioassay Evaluation.

Quorum sensing (QS) is a cell-to-cell communication mechanism that regulates bacterial pathogenicity, biofilm formation, and antibiotic sensitivity. Among the identified quorum sensing, AI-2 QS exists in both Gram-negative and Gram-positive bacteria and is responsible for interspecies communication. Recent studies have highlighted the connection between the phosphotransferase system (PTS) and AI-2 QS, with this link being associated with protein-protein interaction (PPI) between HPr and LsrK. Here, we first discovered several AI-2 QSIs targeting the LsrK/HPr PPI site through molecular dynamics (MD) simulation, virtual screening, and bioassay evaluation. Of the 62 compounds purchased, eight compounds demonstrated significant inhibition in LsrK-based assays and AI-2 QS interference assays. Surface plasmon resonance (SPR) analysis confirmed that the hit compound 4171-0375 specifically bound to the LsrK-N protein (HPr binding domain, KD = 2.51 × 10-5 M), and therefore the LsrK/HPr PPI site. The structure-activity relationships (SARs) emphasized the importance of hydrophobic interactions with the hydrophobic pocket and hydrogen bonds or salt bridges with key residues of LsrK for LsrK/HPr PPI inhibitors. These new AI-2 QSIs, especially 4171-0375, exhibited novel structures, significant LsrK inhibition, and were suitable for structural modification to search for more effective AI-2 QSIs.