Evaluating a Retro-Inverso Type Inhibitor of HTLV-1 Protease as a Competitive Inhibitor.

The inhibition mode of a retro-inverso (RI) inhibitor containing a hydroxyethylamine dipeptide isostere against the human T-cell leukemia virus type-1 (HTLV-1) protease was examined. Enzymatic evaluation of the RI-modified inhibitor containing a D-allo-Ile residue revealed that HTLV-1 was competitively inhibited. IC50 values of the RI-modified inhibitor and pepstatin A, a standard inhibitor of aspartic proteases, were nearly equivalent.

A retro-inverso modified peptide alleviated ovalbumin-induced asthma model by affecting glycerophospholipid and purine metabolism of immune cells.

Allergic asthma is a heterogeneous disease involving a variety of inflammatory cells. Immune imbalance or changes in the immune microenvironment are the essential causes that promote inflammation in allergic asthma. Tetraspanin CD81 can be used as a platform for receptor clustering and signal transmission owing to its special transmembrane structure and is known to participate in the physiological processes of cell proliferation, differentiation, adhesion, and migration. Previous studies have shown that CD81-targeting peptidomimetics exhibit anti-allergic lung inflammation. However, due to the low metabolic stability of peptide drugs, their druggability is limited. Here, we aimed to generate a metabolically stable anti-CD81 peptide, evaluate its anti-inflammatory action and establish its mechanism of action. Based on previous reports, we applied retro-inverse peptide modification to obtain a new compound, PD00 (NH2-D-Gly-D-Ser-D-Thr-D-Tyr-D-Thr-D-Gln-D-Gly-COOH), with high metabolic stability. Enhanced ultraperformance liquid chromatography-tandem mass spectrometry was used to investigate the in vitro and in vivo metabolic stabilities of PD00. The affinities of PD00 and CD81 were studied using molecular docking and surface plasmon resonance techniques. An ovalbumin (OVA)-induced asthma model was used to evaluate the effects of PD00 in vivo. Mice were treated with different concentrations of PD00 (175 and 350 µg/kg) for 10 days. Airway hyperresponsiveness (AHR) to acetyl-ß-methacholine (Mch), inflammatory cell counts in the bronchoalveolar lavage fluid, and serum OVA-specific IgE levels were detected in the mice at the end of the experiment. Lung tissues were collected for haematoxylin and eosin staining, untargeted metabolomic analysis, and single-cell transcriptome sequencing. PD00 has a high affinity for CD81; therefore, administration of PD00 markedly ameliorated AHR and airway inflammation in mice after OVA sensitisation and exposure. Serum OVA-specific IgE levels decreased considerably. In addition, PD00 treatment increased glycerophospholipid and purine metabolism in immune cells. Collectively, PD00 may regulate the glycerophospholipid and purine metabolism pathways to ameliorate the pathophysiological features of asthma. These findings suggest that PD00 is a potential compound for the treatment of asthma.

Design, synthesis and evaluation of 177Lu-labeled inverso and retro-inverso A9 peptide variants targeting HER2-overexpression.

Several HER2-specific peptides are being continuously explored to find a candidate with suitable pharmacokinetic properties for development of effective radiopharmaceutical that can find applications for clinical screening of breast cancer patients. In the present work with an aim of preparing a radiopeptide with improved metabolic stability and in vivo pharmacokinetic performance we modified our previously reported [177Lu]DOTA-L-A9 peptide. Here we designed an 'inverso' peptide with all d-amino acids and a 'retro-inverso' peptide where sequence of d-amino acids was reversed. Higher secondary structure stabilization of retro- inverso A9 variant compared to inverso A9 peptide was evident by circular dichroism studies. The two radiopeptides [177Lu]DOTA-D-A9 and [177Lu]DOTA-rD-A9 exhibited significantly improved in vivo metabolic stability over the original l-peptide. The retro-inverso variant, [177Lu]DOTA-rD-A9 demonstrated better pharmacokinetic behavior with significantly higher tumor uptake than the inverso peptide, [177Lu]DOTA-D-A9 and the original peptide, [177Lu]DOTA-L-A9. In the present case of A9 peptide, reversal of the peptide sequence of d-amino acids boosted the uptake and retention of radioactivity in HER2-positive tumor. The present study can thus guide the design and development of newer and improved versions of peptides.

The Effects of Side-Chain Configurations of a Retro-Inverso-Type Inhibitor on the Human T-Cell Leukemia Virus (HTLV)-1 Protease.

In this study, the effects of side-chain configurations of D-Ile residues of a retro-inverso (RI)-type inhibitor on the human T-cell leukemia virus type 1 (HTLV-1) protease containing a hydroxyethylamine dipeptide isostere were clarified. Prior to evaluation using the RI-type inhibitor, the effects of side-chain configurations of Ile residues of the substrate peptide on the HTLV-1 protease were examined to estimate the influence of side-chain configurations on enzyme activity. Based on the estimation of the influence of side-chain configurations on protease affinity, the RI-type inhibitors containing a D-allo-Ile residue in the corresponding substrate sequence, instead of a D-Ile residue, were synthesized via 9-fluorenylmethoxycarbonyl-based solid-phase peptide synthesis. Refolded recombinant HTLV-1 protease (1-116, L40I) was used for the simple and short evaluation of the inhibitory activities of the synthesized RI-type inhibitors. The results clearly indicated that mimicking the whole topology, comprising both the main- and side-chain structures of the parent inhibitor, is effective for the design of potent RI-modified protease inhibitors.

Recent Applications of Retro-Inverso Peptides.

Natural and de novo designed peptides are gaining an ever-growing interest as drugs against several diseases. Their use is however limited by the intrinsic low bioavailability and poor stability. To overcome these issues retro-inverso analogues have been investigated for decades as more stable surrogates of peptides composed of natural amino acids. Retro-inverso peptides possess reversed sequences and chirality compared to the parent molecules maintaining at the same time an identical array of side chains and in some cases similar structure. The inverted chirality renders them less prone to degradation by endogenous proteases conferring enhanced half-lives and an increased potential as new drugs. However, given their general incapability to adopt the 3D structure of the parent peptides their application should be careful evaluated and investigated case by case. Here, we review the application of retro-inverso peptides in anticancer therapies, in immunology, in neurodegenerative diseases, and as antimicrobials, analyzing pros and cons of this interesting subclass of molecules.

Effect of AmyTrap, an amyloid-ß binding drug, on Aß induced mitochondrial dysfunction and tau phosphorylation in cultured neuroblastoma cells.

Alzheimer's Disease (AD) is the most common cause of dementia, affecting 25 million people worldwide. Accumulation of Amyloid-ß (Aß) in the mitochondria has been shown to adversely affect key enzymes including pyruvate dehydrogenase (PDH), succinate dehydrogenase (SDH), oxoglutarate dehydrogenase (OGDH). Accumulation of Aß is also believed to increase Tau expression and pathology. Tau, in its toxic state, results in synaptic damage causing memory and cognitive dysfunction. We are developing a drug to treat AD namely AmyTrap. The active pharmacological ingredient is a retro inverso, Aß-binding peptide which sequesters Aß. We wanted to examine the effect of AmyTrap peptide on Aß-induced mitochondrial dysfunction and Tau phosphorylation. Therefore, we treated SH-SY5Y neuroblastoma cells with wild-type Aß, a mutant AßY10A, AmyTrap peptide (RI-peptide), or Aß and RI-peptide for 72 h. The mutant AßY10A is known to impact the self-aggregating property of Aß as this Tyr10 is essential for self-aggregation. As expected, AßY10A reversed PDH, OGDH and SDH dysfunction to near normal levels. Further, AßY10A successfully reversed Tau phosphorylation, suggesting that Tyr10 is also associated with Aß-induced cytotoxicity. RI-peptide was able to significantly reverse SDH dysfunction with limited effect on PDH and Tau phosphorylation. The findings are suggestive that the Tyr10 on Aß plays a critical role in the self-aggregation. Further studies are warranted to expand these findings.

Effect of Retro-Inverso Isomer of Bradykinin on Size-Dependent Penetration of Blood-Brain Tumor Barrier.

Retro-inverso bradykinin (RI-BK) has better metabolic stability and higher affinity for the BK type 2 (B2) receptor, compared with bradykinin. At low doses, RI-BK can selectively enhance the permeability of the blood-brain tumor barrier (BBTB) without harming normal brain tissue. In this study, gold nanoparticles (GNPs) of size ranging from 5 to 90 nm are synthesized to assess the optimal size of nanocarriers that achieves maximum brain accumulation after the treatment of RI-BK. The ability of the GNPs to cross the BBTB is tested in a rat C6 glioma tumor model. The results of inductively coupled plasma-mass spectrometry and transmission electron microscopy indicate that GNPs with size of 70 nm achieve maximum permeability to the glioma. The present study supports the conclusion that RI-BK can enhance the permeability of BBTB and provides fundamental information for further development of nanomedicines or nanoprobes for glioma therapy.

Targeting VEGF receptors with non-neutralizing cyclopeptides for imaging applications.

Pharmacological strategies aimed at preventing cancer growth are in most cases paralleled by diagnostic investigations for monitoring and prognosticating therapeutic efficacy. A relevant approach in cancer is the suppression of pathological angiogenesis, which is principally driven by vascular endothelial growth factor (VEGF) or closely related factors and by activation of specific receptors, prevailingly VEGFR1 and VEGFR2, set on the surface of endothelial cells. Monitoring the presence of these receptors in vivo is henceforth a way to predict therapy outcome. We have designed small peptides able to bind and possibly antagonize VEGF ligands by targeting VEGF receptors. Peptide systems have been designed to be small, cyclic and to host triplets of residues known to be essential for VEGF receptors recognition and we named them 'mini-factors'. They have been structurally characterized by CD, NMR and molecular dynamics (MD) simulations. Mini-factors do bind with different specificity and affinity VEGF receptors but none blocks receptor activity. Following derivatization with suitable tracers they have been employed as molecular probes for sensing receptors on cell surface without affecting their activity as is usually observed with other binders having neutralizing activity.

Structure Reconstruction of LyP-1: Lc(LyP-1) Coupling by Amide Bond Inspires the Brain Metastatic Tumor Targeted Drug Delivery.

The stability and binding affinity of targeting ligands are very important in active targeting drug delivery. Herein we used LyP-1 peptide as a model peptide to investigate chemical-biology-based strategies in the design of peptide ligands for active targeting. LyP-1 is a short peptide cyclized with a disulfide bond. It can specifically bind to tumor cells and tumor lymphatics through the interaction with cell-surface protein p32/gC1qR. Lc(LyP-1), with a same sequence of LyP-1, is coupled by amide bond. It showed better cellular uptake and stability in blood in our previous research. Further, usually d-peptide demonstrates higher stability than l-peptide, and it may contribute to better active targeting ability in vivo. Herein, we designed a retro-inverso isomer of Lc(LyP-1), termed Dc(LyP-1), expecting to inspire brain metastatic tumor targeted drug delivery. However, although Lc(LyP-1) showed lower stability than Dc(LyP-1) in fresh rat bold serum, both the 4T1 cellular uptake capacity (89.20%) and p32 protein binding affinity (7.39 × 10-6) were significantly higher than those (33.41%, 1.37 × 10-5) of Dc(LyP-1). Further, Lc(LyP-1) modified PEG-PLA micelles displayed much higher in vivo distribution in brain metastatic tumor than Dc(LyP-1). All results suggested that Lc(LyP-1) had a better performance than Dc(LyP-1) in brain metastatic tumor-targeted drug delivery.

A retro-inverso cell-penetrating peptide for siRNA delivery.

BACKGROUND: Small interfering RNAs (siRNAs) are powerful tools to control gene expression. However, due to their poor cellular permeability and stability, their therapeutic development requires a specific delivery system. Among them, cell-penetrating peptides (CPP) have been shown to transfer efficiently siRNA inside the cells. Recently we developed amphipathic peptides able to self-assemble with siRNAs as peptide-based nanoparticles and to transfect them into cells. However, despite the great potential of these drug delivery systems, most of them display a low resistance to proteases. RESULTS: Here, we report the development and characterization of a new CPP named RICK corresponding to the retro-inverso form of the CADY-K peptide. We show that RICK conserves the main biophysical features of its L-parental homologue and keeps the ability to associate with siRNA in stable peptide-based nanoparticles. Moreover the RICK:siRNA self-assembly prevents siRNA degradation and induces inhibition of gene expression. CONCLUSIONS: This new approach consists in a promising strategy for future in vivo application, especially for targeted anticancer treatment (e.g. knock-down of cell cycle proteins). Graphical abstract RICK-based nanoparticles: RICK peptides and siRNA self-assemble in peptide-based nanoparticles to penetrate into the cells and to induce target protein knock-down.

Retro-inverso peptide inhibitor nanoparticles as potent inhibitors of aggregation of the Alzheimer's Aß peptide.

Aggregation of amyloid-ß peptide (Aß) is a key event in the pathogenesis of Alzheimer's disease (AD). We investigated the effects of nanoliposomes decorated with the retro-inverso peptide RI-OR2-TAT (Ac-rGffvlkGrrrrqrrkkrGy-NH2) on the aggregation and toxicity of Aß. Remarkably low concentrations of these peptide inhibitor nanoparticles (PINPs) were required to inhibit the formation of Aß oligomers and fibrils in vitro, with 50% inhibition occurring at a molar ratio of ~1:2000 of liposome-bound RI-OR2-TAT to Aß. PINPs also bound to Aß with high affinity (Kd=13.2-50 nM), rescued SHSY-5Y cells from the toxic effect of pre-aggregated Aß, crossed an in vitro blood-brain barrier model (hCMEC/D3 cell monolayer), entered the brains of C57 BL/6 mice, and protected against memory loss in APPSWE transgenic mice in a novel object recognition test. As the most potent aggregation inhibitor that we have tested so far, we propose to develop PINPs as a potential disease-modifying treatment for AD.

D-Peptides as Recognition Molecules and Therapeutic Agents.

Over recent years, D-peptides have attracted increasing attention. D-peptides increase enzymatic stability, prolong the plasma half-life, improve oral bioavailability, and enhance binding activity and specificity with receptor or target proteins, in comparison with the corresponding L-peptide. Therefore, D-peptides are considered to have potential as recognition molecules and therapeutic agents. This review focuses on the design and application of D-peptides with biological activity.

Integrin αvß3 targeting activity study of different retro-inverso sequences of RGD and their potentiality in the designing of tumor targeting peptides.

Retro-inverso peptide represented the isomer of a parent peptide in which the direction of the sequence was reversed and the chirality of each amino acid residue was inverted. Generally, retro-inverso peptides possessed equal or even higher activities compared to the original peptide. RGD was a commonly used ligand for tumor and vascular targeting due to its affinity to integrin αvß3 receptors. The biological activity study of the isomers of RGD would indeed provide useful suggestions for the design of tumor targeting peptides. Therefore, the tumor targeting activities of octa-arginine which was modified with different retro-inverso sequences of RGD peptide were investigated in this study. Three different tandem peptides (R8-GDGR, R8-GdGr and R8-GdGR) were designed on the basis of R8-GRGD. The tumor targeting activities of these tandem peptides were evaluated both in vitro and in vivo. Finally, R8-GdGR displayed selective binding affinity to integrin αvß3 at the cellular level, and exhibited efficient tumor homing and penetrating capabilities in vivo. Meanwhile, R8-GdGR also showed stronger neovessel targeting ability compared to the others. In conclusion, all the results demonstrated that dGR possessed similar biological activity to RGD and was a potential ligand for further designing of tumor targeting peptides.

Conformational and biological properties of Bauhinia bauhinioides kallikrein inhibitor fragments with bradykinin-like activities.

Proteinase inhibitors extracted form medicinal plants are an interesting source of new drugs. Modifications in the structure of some of this kind of macromolecules could also lead to compounds of interesting biological properties. In this work, we synthesized and tested one fragment containing the reactive site of the Bauhinia bauhinioides kallikrein inhibitor (BbKI), denoted BbKI51-62 , and a related analog (P2 ) in which a proline residue was inserted in order to mimic the N-terminal region of the bradykinin molecule. The related retro-inverso counterparts Ri-BbKI51-62 and Ri-P2 were also included. The ability of these peptides to induce contraction of stomach fundus isolated from mouse was evaluated as well as their capability to induce calcium release from a cell culture of smooth muscle from guinea pig ileum. The conformational properties of the peptides were evaluated by circular dichroism and their resistance to enzymatic degradation by exposure to human blood plasma. Our results show that neither the parent BbKI51-62 nor its Ri-BbKI51-62 analog exhibit bradykinin-like activity, although the retro-inverso isomer was resistant to blood plasma degradation. On the other hand, the peptides P2 and Ri-P2 presented contractile activities on gastric smooth muscle from stomach fundus possibly by acting via B-2 receptor. Both compounds also induce calcium release from guinea pig ileum muscle cells in a manner similar to bradykinin. Moreover, both compounds also inhibited porcine pancreatic kallikrein. However, conformational analysis did not reveal any direct correlation between structure and biological effects.

Retro-inverso isomer of Angiopep-2: a stable d-peptide ligand inspires brain-targeted drug delivery.

The blood-brain barrier (BBB) prevents most drugs from reaching the site of central nervous system (CNS) diseases, intensively confining the therapeutic efficiency. Angiopep-2 (here termed (L)Angiopep), which is a 19-mer peptide derived from human Kunitz domain, can trigger transcytosis and traverse the BBB by recognizing low density lipoprotein-related protein 1 (LRP-1) expressed on the brain capillary endothelial cells. Various enzymes in the blood and the BBB, however, present multiple metabolic barriers to peptide-inspired brain-targeted drug delivery. Here we designed a retro-inverso isomer of (L)Angiopep, termed (D)Angiopep, to inspire brain-targeted drug delivery. Both (D)Angiopep and (L)Angiopep displayed high uptake capacity in LRP-1 overexpressed cells, including bEnd.3 and U87 cells. (D)Angiopep demonstrated lower uptake efficiency in both cell lines than did (L)Angiopep, suggestive of lower binding affinity to LRP-1 of the d-peptide. (D)Angiopep was resistant to proteolysis in fresh rat blood serum, while more than 85% of (L)Angiopep disappeared within 2 h. Endocytosed (D)Angiopep and (L)Angiopep were found to be colocalized with lysosomal compartments of bEnd.3 cells, indicating that susceptibility to proteolysis of (L)Angiopep in the BBB may further attenuate its transcytosis efficiency. In vivo, (D)Angiopep modified PEG-DSPE micelles displayed high distribution in normal brain and intracranial glioblastoma. Due to the expression of LRP-1 on the BBB and glioblastoma cells, proteolytically stable (D)Angiopep holds much potential for designing two-order brain tumor targeted delivery systems.

Effect of prime-site sequence of retro-inverso-modified HTLV-1 protease inhibitor.

The effects of additional substituents covering the prime-site of retro-inverso (RI)-modified HTLV-1 protease inhibitors containing a hydroxyethylamine isoster were clarified. Stereo-selective construction of the most potent isoster backbone was achieved by the Evans-aldol reaction. Addition of N-acetylated d-amino acid corresponding to the P2' site gave an RI-modified inhibitor showing superior inhibitory activity to the previous inhibitor. Inhibitory activities of the newly synthesized inhibitors suggest that partially modified RI inhibitors would interact with HTLV-1 protease in the same manner as the parent hydroxyethylamine inhibitor.

Scandium-44 Radiolabeled Peptide and Peptidomimetic Conjugates Targeting Neuropilin-1 Co-Receptor as Potential Tools for Cancer Diagnosis and Anti-Angiogenic Therapy.

Pathological angiogenesis, resulting from an imbalance between anti- and pro-angiogenic factors, plays a pivotal role in tumor growth, development and metastasis. The inhibition of the angiogenesis process by the VEGF/VEGFR-2/NRP-1 pathway raises interest in the search for such interaction inhibitors for the purpose of the early diagnosis and treatment of angiogenesis-dependent diseases. In this work we designed and tested peptide-based radiocompounds that selectively bind to the neuropilin-1 co-receptor and prevent the formation of the pro-angiogenic VEGF-A165/NRP-1 complex. Three biomolecules, A7R and retro-inverso DR7A peptides, and the branched peptidomimetic Lys(hArg)-Dab-Pro-Arg (K4R), conjugated with macrocyclic chelator through two linkers' types, were labeled with theranostic scandium-44 radionuclide, and studied in vitro as potential targeted radiopharmaceuticals. ELISA (enzyme-linked immunosorbent assay) studies showed no negative effect of the introduced biomolecules' changes and high NRP-1 affinity in the case of A7R- and K4R-radiocompounds and a lack affinity for DR7A-radiocompounds. All radiopeptides showed a hydrophilic nature as well as high stability against ligand exchange reactions in cysteine/histidine solutions. Unfortunately, all radiocompounds showed unsatisfactory nano-scale stability in human serum, especially for use as therapeutic radioagents. Further work is ongoing and focused on the search for angiogenesis inhibitors that are more human serum stable.

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.

Bioinformatics procedure for investigating senolytic (anti-aging) agents: A digital signal processing technique.

Objective: Cell growth involves cell division. This stops after reaching a certain limit. Some cells become inactive and unable to undergo apoptosis (programmed cell death). These cells accumulate at sites of tissue damage or disease, thus accelerating aging. They are called senescent cells. Therapeutic interventions that can either eliminate senescent cells (senolytics) or suppress their harmful effects (senomorphics) have been developed. Senescence (aging) is caused by the inter- and intramolecular interactions between the domains of forkhead (FHD) and transactivation (TAD), as well as C-terminal region 3 (CR3) and DNA binding (DBD). On the other hand, anti-senescent/senolytic (anti-aging) activities are achieved by disrupting these interactions with CR3- and forkhead box protein O4 (FOXO4)-based peptides, such as ES2 and DRI, respectively. In this study, we use a computerized procedure based on digital signal processing to systematically analyze the inter-molecular interactions between senolytics and their targets. Methods: Informational spectrum method (ISM) is engaged. Results: We obtained the sequences of the peptides from the interacting proteins of CR3 and FOXO4 and evaluated their ability to disrupt the inter-molecular interactions between FOXO4 and DRI and CR3 and BDB, which are responsible for senescence (aging). Our results show that the peptides have different degrees of senolytic (anti-aging) activity, depending on their affinity for CR3 and BDB, or FOXO4 and DRI. We found that enhanced senescence 2 (ES2) has a higher affinity for CR3 and BDB than FOXO4 and DRI, and that the interaction between CR3 and BDB is crucial for aging. Therefore, ES2 and other CR3-based peptides are more potent senolytics than FOXO4-based peptides. Our findings are consistent with previous studies and reveal new insights into the mechanisms of senescence and senolytics. ES2 is considered the best senolytic candidate, as it is 3-7 times more effective than DRI. We verified that ES2 has a weaker interaction with FOXO4 than CR3. However, the performance of DRI has been noted to depend on its intramolecular interactions and stability. Hence, intramolecular analyses using the digital signal processing-based technique has become very vital and will follow. Conclusion: CR3-based peptides are promising candidates for senolytic therapy. Senolytics are linear chains of amino acids that can target and eliminate senescent cells, which are cells that have stopped dividing and contribute to aging and age-related diseases. By using this proposed, novel computerized technique that is based on digital signal processing, senolytics can be easily analyzed and optimized for their effectiveness and safety. This provides a more rational approach to enhancing our longevity and well-being by offering interventions that can delay or reverse aging and insights that can advance the field of gerontology. This procedure also will compliment other approaches such as molecular stimulation, etc.

GnRH receptor-activating autoantibodies in polycystic ovary syndrome: identification of functional epitopes and development of epitope mimetic inhibitors.

PURPOSE: We have recently demonstrated that gonadotrophin-releasing hormone receptor-activating autoantibodies (GnRHR-AAb) are associated with polycystic ovary syndrome (PCOS). The aim of this study was to map the antigenic epitopes of GnRHR-AAb from PCOS patients, and develop retro-inverso peptide inhibitors that specifically target GnRHR-AAb. METHODS: Serum samples from ten GnRHR-AAb-positive PCOS patients and ten GnRHR-AAb-negative healthy controls were tested. Epitope mapping for GnRHR-AAb was performed using a set of 11 overlapping octapeptides spanning the second extracellular loop of GnRHR. Antibody-blocking effect of the designed retro-inverso peptide inhibitors was evaluated in a cell-based bioassay. RESULTS: Two peptide sequences, FSQCVTHC and HCSFSQWW, were found to react with all PCOS sera, but not with control sera. Two retro-inverso peptides that mimic the identified epitopes, d-CHTVCQSF and d-WWQSFSCH, significantly inhibited PCOS serum IgG-induced GnRHR activation. One of these two peptide inhibitors, d-CHTVCQSF, largely suppressed autoantibody-induced GnRHR activation, suggesting that the epitope sequence FSQCVTHC may be a major functional target of GnRHR-AAb. CONCLUSION: We have identified a dominant functional epitope for GnRHR-AAb associated with PCOS, and demonstrated effective blocking of GnRHR-AAb activity with epitope-mimicking retro-inverso peptide inhibitors. These proteolytically stable decoy peptides may have important therapeutic implications in subjects who harbor these autoantibodies.