PRDX1 Interfering Peptide Disrupts Amino Acids 70-90 of PRDX1 to Inhibit the TLR4/NF-κB Signaling Pathway and Attenuate Neuroinflammation and Ischemic Brain Injury.

Ischemic stroke ranks among the leading causes of death and disability in humans and is accompanied by motor and cognitive impairment. However, the precise mechanisms underlying injury after stroke and effective treatment strategies require further investigation. Peroxiredoxin-1 (PRDX1) triggers an extensive inflammatory cascade that plays a pivotal role in the pathology of ischemic stroke, resulting in severe brain damage from activated microglia. In the present study, we used molecular dynamics simulation and nuclear magnetic resonance to detect the interaction between PRDX1 and a specific interfering peptide. We used behavioral, morphological, and molecular experimental methods to demonstrate the effect of PRDX1-peptide on cerebral ischemia-reperfusion (I/R) in mice and to investigate the related mechanism. We found that PRDX1-peptide bound specifically to PRDX1 and improved motor and cognitive functions in I/R mice. In addition, pretreatment with PRDX1-peptide reduced the infarct area and decreased the number of apoptotic cells in the penumbra. Furthermore, PRDX1-peptide inhibited microglial activation and downregulated proinflammatory cytokines including IL-1ß, IL-6, and TNF-α through inhibition of the TLR4/NF-κB signaling pathway, thereby attenuating ischemic brain injury. Our findings clarify the precise mechanism underlying PRDX1-induced inflammation after ischemic stroke and suggest that the PRDX1-peptide can significantly alleviate the postischemic inflammatory response by interfering with PRDX1 amino acids 70-90 and thereby inhibiting the TLR4/NF-κB signaling pathway. Our study provides a theoretical basis for a new therapeutic strategy to treat ischemic stroke.

Inhibition of the RLR signaling pathway by SARS-CoV-2 ORF7b is mediated by MAVS and abrogated by ORF7b-homologous interfering peptide.

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and characterized by dysregulated immune response. Studies have shown that the SARS-CoV-2 accessory protein ORF7b induces host cell apoptosis through the tumor necrosis factor alpha (TNF-α) pathway and blocks the production of interferon beta (IFN-ß). The underlying mechanism remains to be investigated. In this study, we found that ORF7b facilitated viral infection and production, and inhibited the RIG-I-like receptor (RLR) signaling pathway through selectively interacting with mitochondrial antiviral-signaling protein (MAVS). MAVS439-466 region and MAVS Lys461 were essential for the physical association between MAVS and ORF7b, and the inhibition of the RLR signaling pathway by ORF7b. MAVSK461/K63 ubiquitination was essential for the RLR signaling regulated by the MAVS-ORF7b complex. ORF7b interfered with the recruitment of tumor necrosis factor receptor-related factor 6 (TRAF6) and the activation of the RLR signaling pathway by MAVS. Furthermore, interfering peptides targeting the ORF7b complex reversed the ORF7b-suppressed MAVS-RLR signaling pathway. The most potent interfering peptide V disrupts the formation of ORF7b tetramers, reverses the levels of the ORF7b-inhibited physical association between MAVS and TRAF6, leading to the suppression of viral growth and infection. Overall, this study provides a mechanism for the suppression of innate immunity by SARS-CoV-2 infection and the mechanism-based approach via interfering peptides to potentially prevent SARS-CoV-2 infection.IMPORTANCEThe pandemic coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and continues to be a threat to public health. It is imperative to understand the biology of SARS-CoV-2 infection and find approaches to prevent SARS-CoV-2 infection and ameliorate COVID-19. Multiple SARS-CoV-2 proteins are known to function on the innate immune response, but the underlying mechanism remains unknown. This study shows that ORF7b inhibits the RIG-I-like receptor (RLR) signaling pathway through the physical association between ORF7b and mitochondrial antiviral-signaling protein (MAVS), impairing the K63-linked MAVS polyubiquitination and its recruitment of tumor necrosis factor receptor-related factor 6 (TRAF6) to MAVS. The most potent interfering peptide V targeting the ORF7b-MAVS complex may reverse the suppression of the MAVS-mediated RLR signaling pathway by ORF7b and prevent viral infection and production. This study may provide new insights into the pathogenic mechanism of SARS-CoV-2 and a strategy to develop new drugs to prevent SARS-CoV-2 infection.

Restoring GABAB receptor expression in the ventral tegmental area of methamphetamine addicted mice inhibits locomotor sensitization and drug seeking behavior.

Repeated exposure to psychostimulants such as methamphetamine (METH) induces neuronal adaptations in the mesocorticolimbic dopamine system, including the ventral tegmental area (VTA). These changes lead to persistently enhanced neuronal activity causing increased dopamine release and addictive phenotypes. A factor contributing to increased dopaminergic activity in this system appears to be reduced GABAB receptor-mediated neuronal inhibition in the VTA. Dephosphorylation of serine 783 (Ser783) of the GABAB2 subunit by protein phosphatase 2A (PP2A) appears to trigger the downregulation GABAB receptors in psychostimulant-addicted rodents. Therefore, preventing the interaction of GABAB receptors with PP2A using an interfering peptide is a promising strategy to restore GABAB receptor-mediated neuronal inhibition. We have previously developed an interfering peptide (PP2A-Pep) that inhibits the GABAB receptors/PP2A interaction and thereby restores receptor expression under pathological conditions. Here, we tested the hypothesis that restoration of GABAB receptor expression in the VTA of METH addicted mice reduce addictive phenotypes. We found that the expression of GABAB receptors was significantly reduced in the VTA and nucleus accumbens but not in the hippocampus and somatosensory cortex of METH-addicted mice. Infusion of PP2A-Pep into the VTA of METH-addicted mice restored GABAB receptor expression in the VTA and inhibited METH-induced locomotor sensitization as assessed in the open field test. Moreover, administration of PP2A-Pep into the VTA also reduced drug seeking behavior in the conditioned place preference test. These observations underscore the importance of VTA GABAB receptors in controlling addictive phenotypes. Furthermore, this study illustrates the value of interfering peptides targeting diseases-related protein-protein interactions as an alternative approach for a potential development of selective therapeutic interventions.

Identification of the functional PD-L1 interface region responsible for PD-1 binding and initiation of PD-1 signaling.

The PD-1/PD-L1 checkpoint pathway is important for regulating immune responses and can be targeted by immunomodulatory drugs to treat a variety of immune disorders. However, the precise protein-protein interactions required for the initiation of PD-1/PD-L1 signaling are currently unknown. Previously, we designed a series of first-generation PD-1 targeting peptides based on the native interface region of programmed death ligand 1 (PD-L1) that effectively reduced PD-1/PD-L1 binding. In this work, we further characterized the previously identified lead peptide, MN1.1, to identify key PD-1 binding residues and design an optimized peptide, MN1.4. We show MN1.4 is significantly more stable than MN1.1 in serum and retains the ability to block PD-1/PD-L1 complex formation. We further characterized the immunomodulatory effects of MN1.4 treatment by measuring markers of T cell activation in a co-culture model with ovarian cancer cells and peripheral blood mononuclear cells. We found MN1.4 treatment reduced cytokine secretion and suppressed T cell responses in a similar manner as recombinant PD-L1. Therefore, the PD-L1 interface region used to design MN1.4 appeared sufficient to initiate PD-1 signaling and likely represents the minimum necessary region of PD-L1 required for PD-1 recognition. We propose a peptide agonist for PD-1, such as MN1.4, could have several applications for treating autoimmune disorders caused by PD-1 deficiencies such as type 1 diabetes, inflammatory arthritis, or autoimmune side effects arising from monoclonal antibody-based cancer immunotherapies.

The development of an anti-cancer peptide M1-21 targeting transcription factor FOXM1.

BACKGROUND: Transcription factor FOXM1 is a potential target for anti-cancer drug development. An interfering peptide M1-21, targeting FOXM1 and FOXM1-interacting proteins, is developed and its anti-cancer efficacy is evaluated. METHODS: FOXM1 C-terminus-binding peptides are screened by in silico protocols from the peptide library of FOXM1 (1-138aa) and confirmed by cellular experiments. The selected peptide is synthesized into its D-retro-inverso (DRI) form by fusing a TAT cell-penetrating sequence. Anti-cancer activities are evaluated in vitro and in vivo with tumor-grafted nude mice, spontaneous breast cancer mice, and wild-type metastasis-tracing mice. Anti-cancer mechanisms are analyzed. Distribution and safety profiles in mice are evaluated. RESULTS: With improved stability and cell inhibitory activity compared to the parent peptide, M1-21 binds to multiple regions of FOXM1 and interferes with protein-protein interactions between FOXM1 and its various known partner proteins, including PLK1, LIN9 and B-MYB of the MuvB complex, and ß-catenin. Consequently, M1-21 inhibits FOXM1-related transcriptional activities and FOXM1-mediated nuclear importation of ß-catenin and ß-catenin transcriptional activities. M1-21 inhibits multiple types of cancer (20 µM in vitro or 30 mg/kg in vivo) by preventing proliferation, migration, and WNT signaling. Distribution and safety profiles of M1-21 are favorable (broad distribution and > 15 h stability in mice) and the tested non-severely toxic dose reaches 200 mg/kg in mice. M1-21 also has low hemolytic toxicity and immunogenicity in mice. CONCLUSIONS: M1-21 is a promising interfering peptide targeting FOXM1 for the development of anti-cancer drugs.

Targeting the interaction of GABAB receptors with CaMKII with an interfering peptide restores receptor expression after cerebral ischemia and inhibits progressive neuronal death in mouse brain cells and slices.

Cerebral ischemia is the leading cause for long-term disability and mortality in adults due to massive neuronal death. Currently, there is no pharmacological treatment available to limit progressive neuronal death after stroke. A major mechanism causing ischemia-induced neuronal death is the excessive release of glutamate and the associated overexcitation of neurons (excitotoxicity). Normally, GABAB receptors control neuronal excitability in the brain via prolonged inhibition. However, excitotoxic conditions rapidly downregulate GABAB receptors via a CaMKII-mediated mechanism and thereby diminish adequate inhibition that could counteract neuronal overexcitation and neuronal death. To prevent the deleterious downregulation of GABAB receptors, we developed a cell-penetrating synthetic peptide (R1-Pep) that inhibits the interaction of GABAB receptors with CaMKII. Administration of this peptide to cultured cortical neurons exposed to excitotoxic conditions restored cell surface expression and function of GABAB receptors. R1-Pep did not affect CaMKII expression or activity but prevented its T286 autophosphorylation that renders it autonomously and persistently active. Moreover, R1-Pep counteracted the aberrant downregulation of G protein-coupled inwardly rectifying K+ channels and the upregulation of N-type voltage-gated Ca2+ channels, the main effectors of GABAB receptors. The restoration of GABAB receptors activated the Akt survival pathway and inhibited excitotoxic neuronal death with a wide time window in cultured neurons. Restoration of GABAB receptors and neuroprotective activity of R1-Pep was verified by using brain slices prepared from mice after middle cerebral artery occlusion (MCAO). Treatment with R1-Pep restored normal GABAB receptor expression and GABA receptor-mediated K+ channel currents. This reduced MCAO-induced neuronal excitability and inhibited neuronal death. These results support the hypothesis that restoration of GABAB receptor expression under excitatory conditions provides neuroprotection and might be the basis for the development of a selective intervention to inhibit progressive neuronal death after ischemic stroke.

Binding and Kinetic Analysis of Human Protein Phosphatase PP2A Interactions with Caspase 9 Protein and the Interfering Peptide C9h.

The serine/threonine phosphatase PP2A and the cysteine protease Caspase 9 are two proteins involved in physiological and pathological processes, including cancer and apoptosis. We previously demonstrated the interaction between Caspase 9 and PP2A and identified the C9h peptide, corresponding to the binding site of Caspase 9 to PP2A. This interfering peptide can modulate Caspase 9/PP2A interaction leading to a strong therapeutic effect in vitro and in vivo in mouse models of tumor progression. In this manuscript, we investigate (I) the peptide binding to PP2A combining docking with molecular dynamics and (II) the secondary structure of the peptide using CD spectroscopy. Additionally, we compare the binding affinity, using biolayer interferometry, of the wild-type protein PP2A with Caspase 9 and vice versa to that observed between the PP2A protein and the interfering peptide C9h. This result strongly encourages the use of peptides as new therapeutics against cancer, as shown for the C9h peptide already in clinical trial.

Protein phosphatase 2A regulation of GABA B receptors normalizes ischemia-induced aberrant receptor trafficking and provides neuroprotection.

One major factor regulating the strength of GABA B receptor signaling and thereby neuronal excitability is the dynamic control of their cell surface expression. GABA B receptors are constitutively internalized and recycled back to the plasma membrane to maintain a stable number of receptors at cell surface for appropriate signaling. Protein phosphatase 2A (PP2A) dependent dephosphorylation of serine 783 (S783) in the GABA B2 subunit is a key event for downregulating GABA B receptor cell surface expression particularly under conditions associated with excitotoxicity. Here, we investigated the role of PP2A in regulating GABA B receptor cell surface expression under physiological and excitotoxic conditions. For this purpose, we developed an interfering peptide (PP2A-Pep) that inhibits the interaction of GABA B receptors with PP2A. Using cultured cortical neurons, we found that PP2A downregulates GABA B receptor cell surface expression by inhibiting recycling of the receptors and thereby promoting degradation of the receptors. Inhibition of the GABA B receptor/PP2A interaction by PP2A-Pep in cultured cortical neurons restored GABA B receptor cell surface expression after excitotoxic stress and inhibited progressing neuronal death even when added 48 h after the insult. To explore the therapeutic potential of PP2A-Pep, we further analyzed effect of PP2A-Pep in the middle cerebral artery occlusion (MCAO) mouse model of cerebral ischemia. Incubation of brain slices prepared from MCAO-treated mice with PP2A-Pep restored normal GABA B receptor expression and GABA B receptor-mediated inhibition, reduced ischemic-induced overexcitability of neurons, and prevented neuronal death in the ischemic penumbra. This data illustrates the crucial role of regulating GABA B receptor phosphorylation by PP2A for controlling neuronal inhibition and excitability. The results further suggest that interfering with the GABA B receptor/PP2A interaction is a promising strategy for the development of specific therapeutic interventions to treat neurological diseases associated with a disturbed excitation/inhibition balance and downregulation of GABA B receptors.

Development of a novel peptide to prevent entry of SARS-CoV-2 into lung and olfactory bulb cells of hACE2 expressing mice.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus that has caused a global pandemic Coronavirus Disease 2019 (COVID-19). Currently, there are no effective treatments specifically for COVID-19 infection. The initial step in SARS-CoV-2 infection is attachment to the angiotensin-converting enzyme 2 (ACE2) on the cell surface. We have developed a protein peptide that effectively disrupts the binding between the SARS-CoV-2 spike protein and ACE2. When delivered by nasal spray, our peptide prevents SARS-CoV-2 spike protein from entering lung and olfactory bulb cells of mice expressing human ACE2. Our peptide represents a potential novel treatment and prophylaxis against COVID-19.

Targeting the Interaction of GABAB Receptors With CHOP After an Ischemic Insult Restores Receptor Expression and Inhibits Progressive Neuronal Death.

GABAB receptors control neuronal excitability via slow and prolonged inhibition in the central nervous system. One important function of GABAB receptors under physiological condition is to prevent neurons from shifting into an overexcitation state which can lead to excitotoxic death. However, under ischemic conditions, GABAB receptors are downregulated, fostering over-excitation and excitotoxicity. One mechanism downregulating GABAB receptors is mediated via the interaction with the endoplasmic reticulum (ER) stress-induced transcription factor CHOP. In this study, we investigated the hypothesis that preventing the interaction of CHOP with GABAB receptors after an ischemic insult restores normal expression of GABAB receptors and reduces neuronal death. For this, we designed an interfering peptide (R2-Pep) that restored the CHOP-induced downregulation of cell surface GABAB receptors in cultured cortical neurons subjected to oxygen and glucose deprivation (OGD). Administration of R2-Pep after OGD restored normal cell surface expression of GABAB receptors as well as GABAB receptor-mediated inhibition. As a result, R2-Pep reduced enhanced neuronal activity and inhibited progressive neuronal death in OGD stressed cultures. Thus, targeting diseases relevant protein-protein interactions might be a promising strategy for developing highly specific novel therapeutics.

Small Hexokinase 1 Peptide against Toxic SOD1 G93A Mitochondrial Accumulation in ALS Rescues the ATP-Related Respiration.

Mutations in Cu/Zn Superoxide Dismutase (SOD1) gene represent one of the most common causes of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder that specifically affects motor neurons (MNs). The dismutase-active SOD1 G93A mutant is responsible for the formation of toxic aggregates onto the mitochondrial surface, using the Voltage-Dependent Anion Channel 1 (VDAC1) as an anchor point to the organelle. VDAC1 is the master regulator of cellular bioenergetics and by binding to hexokinases (HKs) it controls apoptosis. In ALS, however, SOD1 G93A impairs VDAC1 activity and displaces HK1 from mitochondria, promoting organelle dysfunction, and cell death. Using an ALS cell model, we demonstrate that a small synthetic peptide derived from the HK1 sequence (NHK1) recovers the cell viability in a dose-response manner and the defective mitochondrial respiration profile relative to the ADP phosphorylation. This correlates with an unexpected increase of VDAC1 expression and a reduction of SOD1 mutant accumulation at the mitochondrial level. Overall, our findings provide important new insights into the development of therapeutic molecules to fight ALS and help to better define the link between altered mitochondrial metabolism and MNs death in the disease.

Delivery of the VIVIT Peptide to Human Glioma Cells to Interfere with Calcineurin-NFAT Signaling.

The activation of NFAT (nuclear factor of activated T cells) transcription factors by calcium-dependent phosphatase calcineurin is a key step in controlling T cell activation and plays a vital role during carcinogenesis. NFATs are overexpressed in many cancers, including the most common primary brain tumor, gliomas. In the present study, we demonstrate the expression of NFATs and NFAT-driven transcription in several human glioma cells. We used a VIVIT peptide for interference in calcineurin binding to NFAT via a conserved PxIxIT motif. VIVIT was expressed as a fusion protein with a green fluorescent protein (VIVIT-GFP) or conjugated to cell-penetrating peptides (CPP), Sim-2 or 11R. We analyzed the NFAT expression, phosphorylation, subcellular localization and their transcriptional activity in cells treated with peptides. Overexpression of VIVIT-GFP decreased the NFAT-driven activity and inhibited the transcription of endogenous NFAT-target genes. These effects were not reproduced with synthetic peptides: Sim2-VIVIT did not show any activity, and 11R-VIVIT did not inhibit NFAT signaling in glioma cells. The presence of two calcineurin docking sites in NFATc3 might require dual-specificity blocking peptides. The cell-penetrating peptides Sim-2 or 11R linked to VIVIT did not improve its action making it unsuitable for evaluating NFAT dependent events in glioma cells with high expression of NFATc3.

Pepscan Approach for the Identification of Protein-Protein Interfaces: Lessons from Experiment.

PEPscan is an old approach that has recently gained renewed interest for the identification of interfering peptides (IPs), i.e., peptides able to interfere with protein-protein interactions (PPIs). Its principle is to slice a protein sequence as a series of short overlapping peptides that are synthesized on a peptide array and tested for their ability to bind a partner, with positive spots corresponding to candidate IPs. PEPscan has been applied with a rather large success in various contexts, but the structural determinants underlying this success remain obscure. Here, we analyze the results of 14 PEPscan experiments, and confront the in vitro results with the available structural information. PEPscan identifies candidate IPs in limited numbers that in all cases correspond to solvent-accessible regions of the structures, their location at the protein-protein interface remaining to be further demonstrated. A strong point of PEPscan seems to be its ability to identify specific IPs. IPs identified from the same protein differ depending on the target PPI, and correspond to patches not frequently involved in the interactions seen in the 3D structures available. Overall, PEPscan seems to provide a cheap and rapid manner to identify candidate IPs, that also comes with room for improvement.

Structure-based optimization of a PDZ-binding motif within a viral peptide stimulates neurite outgrowth.

Protection of neuronal homeostasis is a major goal in the management of neurodegenerative diseases. Microtubule-associated Ser/Thr kinase 2 (MAST2) inhibits neurite outgrowth, and its inhibition therefore represents a potential therapeutic strategy. We previously reported that a viral protein (G-protein from rabies virus) capable of interfering with protein-protein interactions between the PDZ domain of MAST2 and the C-terminal moieties of its cellular partners counteracts MAST2-mediated suppression of neurite outgrowth. Here, we designed peptides derived from the native viral protein to increase the affinity of these peptides for the MAST2-PDZ domain. Our strategy involved modifying the length and flexibility of the noninteracting sequence linking the two subsites anchoring the peptide to the PDZ domain. Three peptides, Neurovita1 (NV1), NV2, and NV3, were selected, and we found that they all had increased affinities for the MAST2-PDZ domain, with Kd values decreasing from 1300 to 60 nm, while target selectivity was maintained. A parallel biological assay evaluating neurite extension and branching in cell cultures revealed that the NV peptides gradually improved neural activity, with the efficacies of these peptides for stimulating neurite outgrowth mirroring their affinities for MAST2-PDZ. We also show that NVs can be delivered into the cytoplasm of neurons as a gene or peptide. In summary, our findings indicate that virus-derived peptides targeted to MAST2-PDZ stimulate neurite outgrowth in several neuron types, opening up promising avenues for potentially using NVs in the management of neurodegenerative diseases.

AKAP signaling complexes: pointing towards the next generation of therapeutic targets?

A-kinase anchoring proteins (AKAPs) streamline signal transduction by localizing signaling enzymes with their substrates. Great strides have been made in elucidating the role of these macromolecular signaling complexes as new binding partners and novel AKAPs are continually being uncovered. The mechanics and dynamics of these multi-enzyme assemblies suggest that AKAP complexes are viable targets for therapeutic intervention. This review will highlight recent advances in AKAP research focusing on local signaling events that are perturbed in disease.