Binding Dynamics of a Stapled Peptide Targeting the Transcription Factor NF-Y.

Transcription factors (TFs) play a central role in gene regulation, and their malfunction can result in a plethora of severe diseases. TFs are therefore interesting therapeutic targets, but their involvement in protein-protein interaction networks and the frequent lack of well-defined binding pockets render them challenging targets for classical small molecules. As an alternative, peptide-based scaffolds have proven useful, in particular with an α-helical active conformation. Peptide-based strategies often require extensive structural optimization efforts, which could benefit from a more detailed understanding of the dynamics in inhibitor/protein interactions. In this study, we investigate how truncated stapled α-helical peptides interact with the transcription factor Nuclear Factor-Y (NF-Y). We identified a 13-mer minimal binding core region, for which two crystal structures with an altered C-terminal peptide conformation when bound to NF-Y were obtained. Subsequent molecular dynamics simulations confirmed that the C-terminal part of the stapled peptide is indeed relatively flexible while still showing defined interactions with NF-Y. Our findings highlight the importance of flexibility in the bound state of peptides, which can contribute to overall binding affinity.

Composition and Conformation of Hetero- versus Homo-Fluorinated Triazolamers Influence their Activity on Islet Amyloid Polypeptide Aggregation.

Novel fluorinated foldamers based on aminomethyl-1,4-triazolyl-difluoroacetic acid (1,4-Tz-CF2) units were synthesized and their conformational behaviour was studied by NMR and molecular dynamics. Their activity on the aggregation of the human islet amyloid polypeptide (hIAPP) amyloid protein was evaluated by fluorescence spectroscopy and mass spectrometry. The fluorine labelling of these foldamers allowed the analysis of their interaction with the target protein. We demonstrated that the preferred extended conformation of homotriazolamers of 1,4-Tz-CF2 unit increases the aggregation of hIAPP, while the hairpin-like conformation of more flexible heterotriazolamers containing two 1,4-Tz-CF2 units mixed with natural amino acids from the hIAPP sequence reduces it, and more efficiently than the parent natural peptide. The longer heterotriazolamers having three 1,4-Tz-CF2 units adopting more folded hairpin-like and ladder-like structures similar to short multi-stranded ß-sheets have no effect. This work demonstrates that a good balance between the structuring and flexibility of these foldamers is necessary to allow efficient interaction with the target protein.

Exploiting high-energy hydration sites for the discovery of potent peptide aldehyde inhibitors of the SARS-CoV-2 main protease with cellular antiviral activity.

Small-molecule antivirals that prevent the replication of the SARS-CoV-2 virus by blocking the enzymatic activity of its main protease (Mpro) are and will be a tenet of pandemic preparedness. However, the peptidic nature of such compounds often precludes the design of compounds within favorable physical property ranges, limiting cellular activity. Here we describe the discovery of peptide aldehyde Mpro inhibitors with potent enzymatic and cellular antiviral activity. This structure-activity relationship (SAR) exploration was guided by the use of calculated hydration site thermodynamic maps (WaterMap) to drive potency via displacement of waters from high-energy sites. Thousands of diverse compounds were designed to target these high-energy hydration sites and then prioritized for synthesis by physics- and structure-based Free-Energy Perturbation (FEP+) simulations, which accurately predicted biochemical potencies. This approach ultimately led to the rapid discovery of lead compounds with unique SAR that exhibited potent enzymatic and cellular activity with excellent pan-coronavirus coverage.

Design, synthesis and evaluation of bioactivity of peptidomimetics based on chloroalkene dipeptide isosteres.

Ample biologically active peptides have been found, identified and modified for use in drug discovery to date. However, several factors, such as low metabolic stability due to proteolysis and non-specific interactions with multiple off-target molecules, might limit the therapeutic use of peptides. To enhance the stability and/or bioactivity of peptides, the development of "peptidomimetics," which mimick peptide molecules, is considered to be idealistic. Hence, chloroalkene dipeptide isosteres (CADIs) was designed, and their synthetic methods have been developed by us. Briefly, in a CADI an amide bond in peptides is replaced with a chloroalkene structure. CADIs might be superior mimetics of amide bonds because the Van der Waals radii (VDR) and the electronegativity value of a chlorine atom are close to those of the replaced oxygen atom. By a developed method of the "liner synthesis", N-tert-butylsulfonyl protected CADIs can be synthesized via a key reaction involving diastereoselective allylic alkylation using organocopper reagents. On the other hand, by a developed method of the "convergent synthesis", N-fluorenylmethoxycarbonyl (Fmoc)-protected carboxylic acids can be also constructed based on N- and C-terminal analogues from corresponding amino acid starting materials via an Evans syn aldol reaction and the Ichikawa allylcyanate rearrangement reaction involving a [3.3] sigmatropic rearrangement. Notably, CADIs can also be applied for Fmoc-based solid-phase peptide synthesis and therefore introduced into bioactive peptides including as the Arg-Gly-Asp (RGD) peptide and the amyloid ß fragment Lys-Leu-Val-Phe-Phe (KLVFF) peptide, which are correlated with cell attachment and Alzheimer's disease (AD), respectively. These CADI-containing peptidomimetics stabilized the conformation and enhanced the potency of the cyclic RGD peptide and the cyclic KLVFF peptide.

A virtual library of small molecules mimicking dipeptides.

Virtual combinatorial libraries are prevalent in drug discovery due to improvements in the prediction of synthetic reactions that can be performed. This has gone hand in hand with the development of virtual screening capabilities to effectively screen the large chemical spaces spanned by exhaustive enumeration of reaction products. In this study, we generated a small-molecule dipeptide mimic library to target proteins binding small peptides. The library was created based on the general idea of peptide synthesis, that is, amino acid mimics were reacted in silico to form the dipeptide mimics, yielding 2,036,819 unique compounds. After docking calculations, two compounds from the library were synthesized and tested against WD repeat-containing protein 5 (WDR5) and histamine receptors H1-H4 to evaluate whether these molecules are viable in assays. The compounds showed the highest potency at the histamine H3 receptor, with Ki values in the two-digit micromolar range.

Cyclobutanone Inhibitors of Diaminopimelate Desuccinylase (DapE) as Potential New Antibiotics.

Based on our previous success in using cyclobutanone derivatives as enzyme inhibitors, we have designed and prepared a 37-member library of α-aminocyclobutanone amides and sulfonamides, screened for inhibition of the bacterial enzyme diaminopimelate desuccinylase (DapE), which is a promising antibiotic target, and identified several inhibitors with micromolar inhibitory potency. Molecular docking suggests binding of the deprotonated hydrate of the strained cyclobutanone, and thermal shift analysis with the most potent inhibitor (3y, IC50 = 23.1 µM) enabled determination of a Ki value of 10.2 +/- 0.26 µM and observed two separate Tm values for H. influenzae DapE (HiDapE).

Ligand-Based Design of Selective Peptidomimetic uPA and TMPRSS2 Inhibitors with Arg Bioisosteres.

Trypsin-like serine proteases are involved in many important physiological processes like blood coagulation and remodeling of the extracellular matrix. On the other hand, they are also associated with pathological conditions. The urokinase-pwlasminogen activator (uPA), which is involved in tissue remodeling, can increase the metastatic behavior of various cancer types when overexpressed and dysregulated. Another member of this protease class that received attention during the SARS-CoV 2 pandemic is TMPRSS2. It is a transmembrane serine protease, which enables cell entry of the coronavirus by processing its spike protein. A variety of different inhibitors have been published against both proteases. However, the selectivity over other trypsin-like serine proteases remains a major challenge. In the current study, we replaced the arginine moiety at the P1 site of peptidomimetic inhibitors with different bioisosteres. Enzyme inhibition studies revealed that the phenylguanidine moiety in the P1 site led to strong affinity for TMPRSS2, whereas the cyclohexylguanidine derivate potently inhibited uPA. Both inhibitors exhibited high selectivity over other structurally similar and physiologically important proteases.

Stereochemical Behavior of Pyrrolo-Pyrazole Peptidomimetics Promoting Phase-Selective Supramolecular Organogels.

Supramolecular gels were developed by taking advantage of an assembly of small dipeptides containing pyrrolo-pyrazole scaffolds. The dipeptides were prepared through a robust and ecofriendly synthetic approach from the commercially available starting materials of diazoalkanes and maleimides. By playing with the functionalization of the scaffold, the choice of the natural amino acid, and the stereochemistry, we were able to obtain phase-selective gels. In particular, one peptidomimetic showed gelation ability and thermoreversibility in aromatic solvents at very low concentrations. Rheology tests showed a typical viscoelastic solid profile, indicating the formation of strong gels that were stable under high mechanical deformation. NMR studies were performed, allowing us to determine the conformational and stereochemical features at the base of the supramolecular interactions.

Advancing Cancer Theranostics Through Integrin αVß3-Targeted Peptidomimetic IAC: From Bench to Bedside.

Introduction: The expression of alpha-five beta-three (αVß3) integrins is upregulated in various malignancies undergoing angiogenesis. The development of integrin antagonists as diagnostic probes makes the αVß3 integrin a suitable candidate for targeting tumor angiogenesis. The goal of this study was to optimize the radiolabeling and evaluate the potential of conjugated integrin antagonist carbamate (IAC), a peptidomimetic, as a theranostic radiopharmaceutical for targeting tumor angiogenesis. Methodology: Radiolabeling of DOTAGA [2,2',2" -{10-(2,6-dioxotetrahydro-2H-pyran-3-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl} triacetic-acid]-IAC with [68Ga]Ga, [177Lu]Lu, and [225Ac]Ac was optimized. The binding affinity (Kd) of DOTAGA-IAC for the αVß3 receptor and cancer cell lines was quantified. The biodistribution studies were conducted in healthy Wistar rats. Dosimetry analysis was performed on [177Lu]Lu-DOTAGA-IAC distribution data. A pilot study of [68Ga]Ga-DOTAGA-IAC and [18F]FDG Positron Emission Tomography (PET/CT) imaging was performed in five patients with histopathologically confirmed breast cancer. PET/CT findings were compared between [68Ga]Ga-DOTAGA-IAC and [18F]FDG in these patients. Results: Radiopharmaceuticals were prepared with high radiochemical purity (>99.9%). Kd and Bmax measurements were 15.02 nM and 417 fmol for αVß3 receptor protein: 115.7 nM and 295.3 fmol for C6 glioma cells. Biodistribution studies in rats suggested the excretion via kidneys and partially through the hepatobiliary route. The effective dose of [177Lu]Lu-DOTAGA-IAC was found to be 0.17 mSv/MBq. The dynamic study in patients revealed the optimal imaging time to be 30-35 mins postadministration. Out of the cohort, [68Ga]Ga-DOTAGA-IAC detected the primary lesions in all five patients with a mean standard uptake value (SUVmax) of 3.94 ± 0.58 compared with [18F]FDG (SUVmax 13.8 ± 6.53). Conclusion: The study demonstrates that DOTAGA-IAC exhibits strong binding to αVß3 integrin, positioning it as a promising PET agent for assessing primary and metastatic cancers. The outcomes from the pilot study suggest the potential of [68Ga]Ga-DOTAGA-IAC PET/CT in breast carcinoma diagnosis. While recognizing the theranostic potential of DOTAGA-IAC for αVß3 integrin-expressing tumors, further clinical investigations are warranted to comprehensively assess therapeutic efficacy.

Polymers of functionalized diaminopropionic acid are efficient mediators of active exogenous enzyme delivery into cells.

Delivery of active protein especially enzyme is one of the major therapeutic challenge. Replacing or substituted invalid/improper acting protein offer fast and effective treatment of disease. Herein, we describe the synthesis and properties of biotinylated peptidomimetics consisting of oxoacid-modified 2,3, L-diaminopropionic acid residues with guanidine groups on its side chains. Electrophoretic analysis showed that the obtained compounds interact with FITC-labeled streptavidin or a streptavidin-ß-galactosidase hybrid in an efficient manner. Complexes formed by the abovementioned molecules are able to cross the cell membranes of cancer or healthy cells and show promising compatibility with live cells. Analysis of ß-galactosidase activity inside the cells revealed surprisingly high levels of active enzyme in complex-treated cells compared to controls. This observation was confirmed by immunochemical studies in which the presence of ß-galactosidase was detected in the membrane and vesicles of the cells.

A Novel Peptidomimetic Insecticide: Dippu-AstR-Based Rational Design and Biological Activity of Allatostatin Analogs.

Insect neuropeptides play an essential role in regulating growth, development, reproduction, nerve conduction, metabolism, and behavior in insects; therefore, G protein-coupled receptors of neuropeptides are considered important targets for designing green insecticides. Cockroach-type allatostatins (ASTs) (FGLamides allatostatins) are important insect neuropeptides in Diploptera punctata that inhibit juvenile hormone (JH) synthesis in the corpora allata and affect growth, development, and reproduction of insects. Therefore, the pursuit of novel insecticides targeting the allatostatin receptor (AstR) holds significant importance. Previously, we identified an AST analogue, H17, as a promising candidate for pest control. Herein, we first modeled the 3D structure of AstR in D. punctata (Dippu-AstR) and predicted the binding mode of H17 with Dippu-AstR to study the critical interactions and residues favorable to its bioactivity. Based on this binding mode, we designed and synthesized a series of H17 derivatives and assessed their insecticidal activity against D. punctata. Among them, compound Q6 showed higher insecticidal activity than H17 against D. punctata by inhibiting JH biosynthesis, indicating that Q6 is a potential candidate for a novel insect growth regulator (IGR)-based insecticide. Moreover, Q6 exhibited insecticidal activity against Plutella xylostella, indicating that these AST analogs may have a wider insecticidal spectrum. The underlying mechanisms and molecular conformations mediating the interactions of Q6 with Dippu-AstR were explored to understand its effects on the bioactivity. The present work clarifies how a target-based strategy facilitates the discovery of new peptide mimics with better bioactivity, enabling improved IGR-based insecticide potency in sustainable agriculture.

Effects of alloferon and its analogues on reproduction and development of the Tenebrio molitor beetle.

As the most numerous group of animals on Earth, insects are found in almost every ecosystem. Their useful role in the environment is priceless; however, for humans, their presence may be considered negative or even harmful. For years, people have been trying to control the number of pests by using synthetic insecticides, which eventually causes an increased level of resistance to applied compounds. The effects of synthetic insecticides have encouraged researchers to search for alternatives and thus develop safe compounds with high specificity. Using knowledge about the physiology of insects and the functionality of compounds of insect origin, a new class of bioinsecticides called peptidomimetics, which are appropriately modified insect analogues, was created. One promising compound that might be successfully modified is the thirteen amino acid peptide alloferon (HGVSGHGQHGVHG), which is obtained from the hemolymph of the blue blowfly Calliphora vicinia. Our research aimed to understand the physiological properties of alloferon and the activity of its peptidomimetics, which will provide the possibility of using alloferon or its analogues in the pharmaceutical industry, as a drug or adjuvant, or in agriculture as a bioinsecticide. We used alloferon and its three peptidomimetics, which are conjugates of the native peptide with three unsaturated fatty acids with various chain lengths: caprylic, myristic, and palmitic. We tested their effects on the morphology and activity of the reproductive system and the embryogenesis of the Tenebrio molitor beetle. We found that the tested compounds influenced the growth and maturation of ovaries and the expression level of the vitellogenin gene. The tested compounds also influenced the process of egg laying, embryogenesis, and offspring hatching, showing that alloferon might be a good peptide for the synthesis of effective bioinsecticides or biopharmaceuticals.

Understanding the Structural Requirements of Peptide-Protein Interaction and Applications for Peptidomimetic Development.

Protein-protein interaction is at the heart of most biological processes, and small peptides that bind to protein binding sites are resourceful tools to explore and understand the structural requirements for these interactions. In that sense, phage display is a well-suited technology to study protein-protein interactions, as it allows for unbiased screening of billions of peptides in search for those that interact with a protein binding domain. Here, we will illustrate how two distinct but complementary approaches, phage display and nuclear magnetic resonance (NMR), can be utilized to unveil structural details of peptide-protein interaction. Finally, knowledge derived from phage mutagenesis and NMR studies can be streamlined for quick peptidomimetic design and synthesis using the retroinversion approach to validate using in vitro and in vivo assays the therapeutic potential of peptides identified by phage display.

Computer-aided molecular design and optimization of potent inhibitors disrupting APC‒Asef interaction.

Colorectal cancer (CRC) is the second leading cause of cancer mortality worldwide. At initial diagnosis, approximately 20% of patients are diagnosed with metastatic CRC (mCRC). Although the APC‒Asef interaction is a well-established target for mCRC therapy, the discovery and development of effective and safe drugs for mCRC patients remains an urgent and challenging endeavor. In this study, we identified a novel structural scaffold based on MAI inhibitors, the first-in-class APC‒Asef inhibitors we reported previously. ONIOM model-driven optimizations of the N-terminal cap and experimental evaluations of inhibitory activity were performed, and 24-fold greater potency was obtained with the best inhibitor compared to the parental compound. In addition, the cocrystal structure validated that the two-layer π‒π stacking interactions were essential for inhibitor stabilization in the bound state. Furthermore, in vitro and in vivo studies have demonstrated that novel inhibitors suppressed lung metastasis in CRC by disrupting the APC‒Asef interaction. These results provide an intrinsic structural basis to further explore drug-like molecules for APC‒Asef-mediated CRC therapy.

Development of Clarstatin, a Novel Drug Lead for the Therapy of Autoimmune Uveitis.

We describe the design, synthesis, and activity of a potent thiourea-bridged backbone cyclic peptidomimetic known as Clarstatin, comprising a 5-amino acid sequence (Q/D)1-(R/K)2-X3-X4-A5-(Gln/Asp)1-(Arg/Lys)2-AA3-AA4-Ala5-based on a motif called "shared epitope (SE)", specifically present in specific alleles of the HLA-DRB1 gene. This SE binds to a particular site within the proline reach domain (P-domain) of the cell surface-calreticulin (CS-CRT). CS-CRT is a multifunctional endoplasmic reticulum (ER) calcium-binding protein that is located on the cell surface of T cells and triggers innate immune signaling, leading to the development of inflammatory autoimmune diseases. The development of Clarstatin was based on the parent peptide W-G-D1-K2-S3-G4-A5- derived from the active region of the SE. Following the design based on the cycloscan method, the synthesis of Clarstatin was performed by the Fmoc solid phase peptide synthesis (SPPS) method, purified by HPLC to 96% homogeneity, and its structure was confirmed by LC-MS. Clarstatin reduced calcium levels in Jurkat lymphocyte cultures, ameliorated uveitis in vivo in the experimental autoimmune uveitis (EAU) mice model, and was safe upon acute toxicity evaluation. These findings identify Clarstatin as a promising lead compound for future drug development as a novel class of therapeutic agents in the therapy of uveitis.

Current and Ongoing Developments in Targeting Clostridioides difficile Infection and Recurrence.

Clostridioides difficile is a Gram-positive, spore-forming anaerobic bacterial pathogen that causes severe gastrointestinal infection in humans. This review provides background information on C. difficile infection and the pathogenesis and toxigenicity of C. difficile. The risk factors, causes, and the problem of recurrence of disease and current therapeutic treatments are also discussed. Recent therapeutic developments are reviewed including small molecules that inhibit toxin formation, disrupt the cell membrane, inhibit the sporulation process, and activate the host immune system in cells. Other treatments discussed include faecal microbiota treatment, antibody-based immunotherapies, probiotics, vaccines, and violet-blue light disinfection.

CD74-AKT Axis Is a Potential Therapeutic Target in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) cells are often resistant to FAS (CD95)-mediated apoptosis, but the underlying molecular mechanism(s) is not fully understood yet. Notably, the expression of the type II transmembrane protein, CD74, is correlated with chemotherapy-resistant and more invasive forms of cancers via unknown mechanisms. Here, we analyzed gene expression pattern of cancer patients and/or patient-derived xenograft (PDX) models and found that mRNA and protein levels of CD74 are highly expressed in TNBC and correlated with cancer stem cells (CSCs) and epithelial-mesenchymal transition (EMT) properties. Mechanistically, we found that AKT activation is likely critical for maintaining CD74 expression and protein stability to favor its oncogenic functions. Physiologically, epidermal growth factor (EGF) along with CD74 could activate AKT signaling, likely through binding of phosphorylated AKT (S473) to CD74, whereas inhibition of AKT could impair stability of CD74. We also revealed that CD74 binds to FAS and interferes with the intrinsic signaling of FAS-mediated apoptosis. As such, selective targeting of the CD74/FAS complex using the AKT inhibitor along with the CD74-derived peptide could synergistically restore and activate FAS-mediated apoptosis. Therefore, our approach of mobilizing apoptosis pathways likely provides a rationale for TNBC treatment by targeting the CD74/FAS and CD74-AKT axes.

Amide-to-Triazole Switch in Somatostatin-14-Based Radioligands: Impact on Receptor Affinity and In Vivo Stability.

The use of metabolically stabilized, radiolabeled somatostatin (SST) analogs ([68Ga]Ga/[177Lu]Lu-DOTA-TATE/TOC/NOC) is well established in nuclear medicine. Despite the pivotal role of these radioligands in the diagnosis and therapy of neuroendocrine tumors (NETs), their inability to interact with all five somatostatin receptors (SST1-5R) limits their clinical potential. [111In]In-AT2S is a radiolabeled DOTA-conjugate derived from the parent peptide SST-14 that exhibits high binding affinity to all SSTR subtypes, but its poor metabolic stability represents a serious disadvantage for clinical use. In order to address this issue, we have replaced strategic trans-amide bonds of [111In]In-AT2S with metabolically stable 1,4-disubstituted 1,2,3-triazole bioisosteres. From the five cyclic triazolo-peptidomimetics investigated, only [111In]In-XG1 combined a preserved nanomolar affinity for the SST1,2,3,5R subtypes in vitro and an improved stability in vivo (up to 17% of intact peptide 5 min postinjection (pi) versus 6% for [111In]In-AT2S). The involvement of neprilysin (NEP) in the metabolism of [111In]In-XG1 was confirmed by coadministration of Entresto®, a registered antihypertensive drug, in vivo releasing the selective and potent NEP-inhibitor sacubitrilat. A pilot SPECT/CT imaging study conducted in mice bearing hSST2R-positive xenografts failed to visualize the xenografts due to the pronounced kidney uptake (>200% injected activity (IA)/g at 4 h pi), likely the result of the formation of cationic metabolites. To corroborate the imaging data, the tumors and the kidneys were excised and analyzed with a γ-counter. Even if receptor-specific tumor uptake for [111In]In-XG1 could be confirmed (1.61% IA/g), further optimization is required to improve its pharmacokinetic properties for radiotracer development.

Conformationally constrained cyclic grafted peptidomimetics targeting protein-protein interactions.

Sunflower trypsin inhibitor-1 (SFTI-1) structure is used for designing grafted peptides as a possible therapeutic agent. The grafted peptide exhibits multiple conformations in solution due to the presence of proline in the structure of the peptide. To lock the grafted peptide into a major conformation in solution, a dibenzofuran moiety (DBF) was incorporated in the peptide backbone structure, replacing the Pro-Pro sequence. NMR studies indicated a major conformation of the grafted peptide in solution. Detailed structural studies suggested that SFTI-DBF adopts a twisted beta-strand structure in solution. The surface plasmon resonance analysis showed that SFTI-DBF binds to CD58 protein. A model for the protein-SFTI-DBF complex was proposed based on docking studies. These studies suggested that SFTI-1 grafted peptide can be used to design stable peptides for therapeutic purposes by grafting organic functional groups and amino acids. However, when a similar strategy was used with another grafted peptide, the resulting peptide did not produce a single major conformation, and its biological activity was lost. Thus, conformational constraints depend on the sequence of amino acids used for SFTI-1 grafting.

Evaluación del efecto in vitro de anticuerpos inducidos por péptido-miméticos derivados del antígeno de superficie de merozoito-2 de plasmodium en malaria causada por plasmodium yoelii y plasmodium berghei en ratones BALB/c / Assessment of the in vitro effect of antibodies induced by peptido-mimetics derived from the Plasmodium merozoite surface protein-2 in malaria caused by P. berghei and P. yoelii in BALB/c mice

Con base en estudios realizados previamente en los cuales se identificaron los residuos críticos para la unión de la secuencia 21KNESKYSNTFINNAYNMSIR40 del antígeno MSP-2 del Plasmodium falciparum, se diseñaron y sintetizaron dos secuencias de pseudopéptidos amida reducida en las cuales se sustituyó un enlace peptídico normal por su isóstero ψ[CH2-NH] entre los residuos fenilalanina-isoleucina y entre los residuos isoleucina-asparagina, para dar lugar a los análogos codificados ψ-128 (forma monomérica), ψ-129 (forma polimérica), ψ-130 (forma monomérica) y ψ-131 (forma polimérica). Con los péptido-miméticos obtenidos en forma de polímero se inmunizaron ratones BALB/c para generar anticuerpos monoclonales que presentaron isotipo IgM. Mediante ensayos controlados de inmunización in vitro se indujo el cambio isotipo de los clones reactivos aislados de los hibridomas obtenidos de manera reproducible. Las inmunoglobulinas aisladas se ensayaron por su capacidad funcional neutralizadora de la infección controlada in vitro de cepas de Plasmodium de roedores a glóbulos rojos. Los resultados obtenidos evidencian el papel que pueden tener los anticuerpos inducidos por péptido-miméticos Plasmodium en ensayos de infección realizados en modelos animales de experimentación.

Based on previous studies in which those residues being critical for Plasmodium falciparum binding to red blood cells (RBCs) through the antigenic sequence (21KNESKYSNTFINNAYNMSIR40) from the merozoite surface antigen-2 (MSP-2) were identified, we have designed and synthesized two reduced amide pseudopeptide sequences based on the 31IN32 binding motif. Synthesized peptidomimetics, possess each one a modified peptide bond that presented as a ψ[CH2-NH] reduced amide isoster bond, to allowing the Phe-Ile modified aminoacid pair allowing pseudopeptides coded ψ-128 (monomer form) and ψ-129 (polymer form) and the Ile-Asn modified aminoacid pair for pseudopeptides coded ψ-130 (monomer form) and ψ-131 (polymer form). By using the polymer forms of both peptido-mimetics as immunogens, monoclonal antibodies were produced in BALB/c mice. These Ig showed an IgM isotype. The isotype antibody switching was lead by in vitro immunization of the original hybridomas. Isolated immunoglobulins were tested for their functional in vitro activity, on a infection controlled experiment of rodent Plasmodium strains infecting red blood cells. Obtained results reveal the role played by antibodies to peptido-mimetic in infection assays performed further on animal experimental models.