Analytical and Bioanalytical Chemistry https://doi.org/10.1007/s00216-021-03424-2 RESEARCH PAPER Design and characterization of high-affinity synthetic peptides as bioreceptors for diagnosis of cutaneous leishmaniasis Y. Andrea Prada 1 & Maria Soler 2,3 & Fanny Guzmán 4 & John J. Castillo 5 & Laura M. Lechuga 2,3 & Enrique Mejía-Ospino 1 Received: 7 April 2021 / Revised: 12 May 2021 / Accepted: 20 May 2021 # Springer-Verlag GmbH Germany, part of Springer Nature 2021 Abstract Cutaneous leishmaniasis (CL) is one of the illnesses caused by Leishmania parasite infection, which can be asymptomatic or severe according to the infecting Leishmania strain. CL is commonly diagnosed by directly detecting the parasites or their DNA in tissue samples. New diagnostic methodologies target specific proteins (biomarkers) secreted by the parasite during the infection process. However, specific bioreceptors for the in vivo or in vitro detection of these novel biomarkers are rather limited in terms of sensitivity and specificity. For this reason, we here introduce three novel peptides as bioreceptors for the highly sensitive and selective identification of acid phosphatase (sAP) and proteophosphoglycan (PPG), which have a crucial role in leishmaniasis infection. These high-affinity peptides have been designed from the conservative domains of the lectin family, holding the ability to interact with the biological target and produce the same effect than the original protein. The synthetic peptides have been characterized and the affinity and kinetic constants for their interaction with the targets (sAP and PPG) have been determined by a surface plasmon resonance biosensor. Values obtained for KD are in the nanomolar range, which is comparable to high-affinity antibodies, with the additional advantage of a high biochemical stability and simpler production. Pep2854 exhibited a high affinity for sAP (KD = 1.48 nM) while Pep2856 had a good affinity for PPG (KD 1.76 nM). This study evidences that these peptidomimetics represent a novel alternative tool to the use of high molecular weight proteins for biorecognition in the diagnostic test and biosensor devices for CL. Keywords Cutaneous leishmaniasis . High-affinity peptides . Lectins . Proteophosphoglycans . Acid phosphatase Introduction Leishmaniasis, caused by Leishmania parasites, is one of the infectious diseases with the highest incidence in the tropical * John J. Castillo regions of South America, Africa, the Mediterranean region, jcasleon@uis.edu.co and Central Asia, affecting nearly 100 countries. According to the World Health Organization (WHO), there are one million 1 Laboratorio de Espectroscopia Atómica y Molecular (LEAM), cases of infection each year, including asymptomatic cases Universidad Industrial de Santander, Carrera 27 Calle 9, which are not reported [1, 2]. Leishmaniasis affects the most Bucaramanga, Santander 678, Colombia vulnerable population in the world, usually associated with 2 Networking Research Center on Bioengineering, Biomaterials and inadequate nutrition, immunosuppression, and deficiency in Nanomedicine (CIBER-BBN), 28029 Madrid, Spain the public health system. There are three clinical manifesta- 3 Nanobiosensors and Bioanalytical Applications Group (NanoB2A), tions of leishmaniasis infection: cutaneous leishmaniasis Catalan Institute of Nanoscience and Nanotechnology (ICN2), CSIC, and BIST, Campus UAB, Bellaterra, 08193 Barcelona, Spain (CL), mucocutaneous leishmaniasis (ML), and visceral leish- 4 maniasis (VL), known as kala-azar [3]. CL is the most fre- Laboratorio de Síntesis de Péptidos, Núcleo de Biotecnología Curauma (NBC), Pontificia Universidad Católica de Valparaíso, quent form of the disease; it causes skin lesions that fortunate- 2373223 Valparaíso, Chile ly can heal without treatment in many cases [4]. ML occurs 5 Grupo de Investigación en Bioquímica y Microbiología (GIBIM), when the parasite spreads into the nose, mouth, and throat Universidad Industrial de Santander, Bucaramanga 680002, producing severe ulcers, destroying the mucous membranes. Colombia It can be cured, but the symptoms might not be detected until Prada Y.A. et al. years after infection and, if not treated, it can evolve into mostly during the infective phase associated to the serious complications [5]. VL is the most lethal form of the promastigote form; therefore, they can play a key critical role disease; it damages internal organs like spleen or liver, and it as clinical targets for both diagnosis and vaccine development affects the bone marrow and the immune system, which re- [21]. sults fatal for the patient and death may occur due to compli- The sAP are complex enzymes, N-carbohydrate and pre- cations associated with secondary infections [6]. dominant O-linked phosphorylated complex enzymes, with To date, there is no vaccine available against leishmaniasis. molecular weight between 90 and 130 kDa, which are consid- However, a recent study predicted the 3D structure of a vac- ered a virulence factors [22]. The sAP inhibits the production cine candidate against VL caused by Leishmania donovani of superoxide anions by neutrophils and macrophages, which based on VC1/VC2 modelled using a molecular dynamics is closely related to the survival of the parasite inside the host coupled TLR-2 and TLR4 receptors [7]. Therefore, an accu- cell. The proteophosphoglycans (PPG), with a molecular rate and early diagnosis of the disease is crucial for the timely weight around 200 kDa, are involved in the mammalian in- administration of treatment and the reduction of the infectious fection and may contribute to the binding of Leishmania to the burden [8, 9]. Importantly, due to the geographic and socio- host cell [23]. logic distribution of leishmaniasis incidence, it is essential to Nowadays, it is well known that the interaction of develop new diagnostic tests that can be implemented as mannose-binding lectin with the specific carbohydrates of point-of-care (POC) devices and applied for routine screening glycoconjugates on the cell surface of Leishmania acts as an analysis [10, 11]. The broad spectrum of clinical manifesta- alternative pathway in the complement activation mechanism tions of CL makes its timely diagnosis complicated. antibody-independent response [24, 25]. In this sense, lectins Currently, standard methods for the diagnosis of CL include can recognize specific glycan arrangements in the PPG mole- microscopy and microbiological culture of the parasite, which cules [26]. The aim of this work is to design, synthesize, and require centralized laboratories, highly specialized personnel, characterize a new peptide-based galactolectins with en- and up to several days for providing conclusive results [12]. hanced robustness and affinities for both sAP and PPG, to The detection of CL via blood serological tests shows a very be used for the highly sensitive detection of leishmaniasis low sensitivity due to a deficient humoral response by the [27]. immune system [13]. This is because the immunological ef- Based on the rational bioinformatic design and theoretical fect in humans is induced by the parasite amastigote form of modelling of the interaction, we have obtained three peptide the parasite (reproductive phase) and not by the promastigote sequences specifically targeting small regions (10–15 amino form (infective phase), which infects the macrophage cells acid residues) of the lectin domains that can keep the immediately after contagion [14]. Clinical assays targeting structure-activity relationship. Peptides were synthesized gene sequences of the parasite using the polymerase chain using Fmoc solid-phase peptide chemistry. Synthetic peptides reaction (PCR) technique have also been developed, as well have been fully characterized to determine the binding affin- as immunochemical tests targeting antigens of the cell mem- ities and kinetic parameters for the interaction with brane, such as Kala-Azar Detect and IT-LEISH for VL, based Leishmania PPG and sAP proteins by surface plasmon reso- on recombinant Leishmania protein K39, and Montenegro nance (SPR) biosensor analysis [28]. SPR is a technique that skin test (MTS) for CL [15, 16]. Other candidates for the plays a prominent role in biomolecular interaction characteri- detection of leishmaniasis are the membrane glycoproteins zation due to its ability to detect and monitor molecular bind- gp63, gp70 and gp72, which are specific for the Leishmania ing events in real time without the use of labels. A home-made genus, and have improved the sensitivity and specificity of SPR biosensor working in Kretschmann configuration was ELISA tests [17]. employed for the analysis and determination of the binding However, these tests are prone to cross-reactions with other affinities and kinetic parameters of the interactions between diseases caused by trypanosome family members. The limita- the target proteins and the new peptides. The working princi- tions of the conventional diagnostic methods are driving re- ple of the SPR biosensor is based on the high sensitivity of an search efforts towards the finding of direct biomarkers and evanescent plasmonic field generated at the interface of a potential targets to increase the sensitivity of the diagnosis nanometric metal layer (i.e. 50 nm of gold) and the dielectric [18]. Recently, secreted proteins are considered an untapped medium when the light strikes at a specific angle. The prop- source of possible antigens and are being exploited using erties of the evanescent field depend significantly on the re- combinations of bioinformatic and immunoproteomics ap- fractive index of the medium, whereby the small changes due proaches [19, 20]. Our focus is directed to the glycoproteins to molecular interactions can be measured, as well as the for- secreted during the infectious process by Leishmania parasite, mation of molecular pairs (target-bioreceptor) that takes place which are promising biomarkers for the detection of CL. on the sensor surface. The SPR analysis requires that one of Among them, the acid phosphatases (sAP) and the studied molecules is immobilized on the gold surface chip. proteophosphoglycans (PPG) are secreted by the parasite Our findings demonstrate the valuable potential of the new Design and characterization of high-affinity synthetic peptides as bioreceptors for diagnosis of cutaneous... synthetic peptides as bioreceptors for the direct assay of leish- algorithms found the binding site with full spatial flexibility maniasis biomarkers in diagnostic tests. The higher affinity of peptide and decreased the fluctuation on the protein back- and stability of our receptors might greatly enhance the ana- bone without an a priori knowledge of the binding site [29, lytical performance of conventional clinical assays or innova- 30]. To evaluate the results, the RMSD value was used, which tive diagnostic devices, such as point-of-care biosensors. is defined as the root mean square deviation of the peptide- protein interaction. For selecting the better models, the contact maps were calculated with a cut-off distance value of 4.5 Å for Materials and methods each peptide-glycoconjugate complex (ESM Fig. S3a–b). General chemistry Synthesis and purification of peptides All common reagents and materials were obtained from a commercial source such as Sigma-Aldrich and MERCK and Pep2854 (HYSRTENMGTSNAC), Pep2855 used without further purification unless otherwise indicated. (KTFLKICKRPYVPSEP), and Pep2856 Glassware was oven-dried before use and the solutions pre- (INKQGDVTVLSNGNTQLT) were synthesized by solid- pared in deionized water (Resistivity 18.2 MΩ). phase peptide synthesis following the Fmoc methodology. Briefly, peptide building blocks were loaded on of Rink amide Bioinformatic peptide design 4MBHA resin (0.55 mmol/g substitution; 100 mg per tea bag) followed by chain linkage via the coupling of Fmoc-protected The protein sequences of secreted acid phosphatase and L-amino acids between the C-terminal residue and the resin. proteophosphoglycans reported by Leishmania ssp. parasites The amino acids were coupled using a mixture of the L-amino were selected on UniProt Data Bank (Available on: www. acid, O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium uniprot.org/). About twelve sequences of sAP and four h e x af l uo r o p ho s p h a t e ( H B T U ) , o r o x i m e an d N - sequences of PPG with up to 60% identity analysed and ethyldiisopropylamine (DIEA) in the ratio 5:5:5:7.5, respec- aligned using both BLAST and Clustal Omega tools tively, for over 3 h. The Fmoc protecting groups were re- (Available on UniProt) for finding a conservative domain in moved with piperidine (20% v/v in 0.01% Tween 20 solu- an independent bioinformatic analysis are reported in the tion). All coupling reactions were carried out in Supplementary Information (ESM) Fig. S1a–b. dimethylformamide. The peptides were cleaved and the lateral Subsequently, the native models for both sAP and PPG con- chain protecting groups were removed using a cocktail con- servative regions were obtained from the GalaxyWEB web taining trifluoroacetic acid (TFA), triisopropylsilane (TIS), server, which allowed making the prediction, refinement, and water (ratio 95:2.5:2.5). Lastly, the peptides were precip- and optimization of the protein structure based on protein itated with cold diethyl ether, centrifuged thrice, and the pellet template by rescoring HHsearch results. Then a group of 15 was dissolved in water before lyophilization. The crude pep- sequences of glycoconjugate-recognized lectins were selected tides were purified by RP-HPLC on a Zorbax C-18 prepara- from Protein Data Bank and ten fragment peptides between 14 tive column (7.0 μm; 21.5 × 250 mm) using a gradient from and 18 amino acid residues of different motif regions of the 30 to 70% of acetonitrile (ACN) (Mobil phase A: H2O, proteins which were extracted from Protein Data Bank 0.001% TFA, and B: ACN, 0.001% TFA). (https://www.rcsb.org/) with ID codes: 1CIW; ICQ9; ICR7; IQF3; 1RIR; 2DHI; 2DV9; 2DVA;2DVB;2DVD; 2DVF; Mass spectrometry analysis 2DVG; 2DVP; 2TEP; 1BZW. The secondary structure conformation of each peptide was modelled and visualized The fractions collected from RP-HPLC were analysed by using the PEPFOLD server and PyMOL v2.3 respectively. MALDI-TOF spectrometry in an UltrafleXtreme Bruker The experimental data suggest that three peptide sequences Daltonics mass spectrometer using a dried-droplet method described as Pep2854, Pep2855, and Pep2856 adopted a using an α-cyano-4-hydroxycinnamic acid saturated solution conformational space-optimized based on high interaction (0.001% TFA in ACN/H2O) as matrix substance for MALDI. similarity with the targets. Theoretical modelling of peptide-glycoprotein Circular dichroism analysis interactions The secondary structure peptide structures were analysed by CABS-dock server (http://biocomp.chem.uw.edu.pl/ circular dichroism in a Jasco J-815 spectropolarimeter with an CABSdock) was used to investigate the protein-peptide inter- acquisition speed of 20 nm/min in a spectral region of 190– action. For the performance of the dynamic simulation, the 260 nm. Prada Y.A. et al. Surface plasmon resonance biosensor Binding interaction analysis on sensor chips SPR experiments were performed at room temperature The dilution series of targets were injected (sAP and PPG) in (25 °C) on a home-made SPR biosensor. The sensor platform separate experiments for each peptide without exceeding the is based on the Kretschmann configuration that allowed the saturation concentration of the analyte onto the sensor chip monitoring of the spectrum of the reflected light at 70° of surface. In the case of sAP, the range of concentrations incident angle on a 50-nm layer of the gold sensor chip, pre- injected was from 5.2 to 104 nM in 10 mM MES buffer viously biofunctionalized with each peptide. The biosensor pH 6.5. Moreover, the PPG was injected in concentrations surface is clamped in a flow cell and is irradiated with a hal- between 3.0 and 58 nM prepared in 10 mM acetate buffer ogen lamp (HL-2000, Ocean Optics, USA) in transverse elec- pH 4.5. The running buffer for each assay was the same as tric polarization mode; the light passes through a trapezoidal for the sample to avoid a bulk refractive index effect, and the prism for plasmonic excitation. Then, the reflected light is flow rate was 30 μL/min applied for 15 min. The regeneration collected by an optical fibre coupled to a CCD spectrometer step was carried out so that all peptides were able to dissociate (Ocean Optics-Jazz Module, USA). The microfluid delivery entirely using 5.0 mM of NaOH at a flow rate of 30 μL/min system includes a syringe pump (New Era, NE1000, USA), for 2 min as shown in Fig. S6 (see ESM). The sensorgrams connected to the flow cell. Samples of sAP and PPG were acquired were fitted to a one-site binding interaction model manually injected using an injection valve (IDEZ Health and using GraphPad Prism software (Version 9.0). This model Science, V-451, USA). Thereby, the biomolecular interac- assumed that the formation of the target-ligand complex is tions that take place on the sensor surface can be detected by not limited by mass transport because the SPR biosensor can the changes in the refraction index and the displacement of the keep a constant target concentration in a continuous flow. plasmon wavelength (Δλ SPR). Data are presented as a Therefore, in the kinetic analysis, the constants of association sensorgram where the ΔλSPR is evaluated in real time. (kon) and dissociation (koff) were calculated from the plot of ΔλSPR vs concentration [28]. Consequently, the equilibrium dissociation constant (KD), known as affinity, was determined Sensor surface biofunctionalization from the relation KD = koff/kon. Additionally, the chi2 value was reported as a parameter indicative of the fitting The sensor chip is based on an ultra-thin gold layer (50 nm confidence. thickness) deposited on a glass slide previously covered For the determination of SPR sensitivity, we used buffer with titanium (2.0 nm thickness). The sensor chips were PBS serial solutions prepared in Milli-Q water; then refraction washed with acetone, isopropanol, and Milli-Q water by index of each PBS solution was measured in a refractometer immersing at boiling temperature. After that, they were and was injected in order of less to major concentration. Then dried with N2 and placed in an ozone cleaner (BioForce Δλ was determined from the LSPR vs real-time graphical as Nanoscience, USA) for 40 min. A self-assembled mono- shown in the Fig. S4 of ESM. layer (SAM) was carried out ex situ using a mixture of functional alkanethiols, mercaptohexadecanoic acid (MHDA), and mercaptoundecanol (MUOH) in a 1:1 ratio Results and analysis and total concentration of 250 μM in ethanol, where the sensor chips were incubated overnight. Then, the Design and synthesis of specific PPG and sAP nanoplasmonic sensors were rinsed with ethanol and dried bioreceptor peptides with N2. The carboxylic group activation was performed via carbodiimide ester formation using a mixture of 1-eth- We have developed three new synthetic peptides for the sen- yl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N- sitive and selective detection of PPC and sAP proteins, secret- hydroxysuccinimide ester (NHS) (0.2 M/0.05 M) solution ed by the Leishmania parasite during the infection process. in 2-(N-morpholino)ethanesulfonic acid (MES) buffer The rational design of peptides was based on the conservative (100 mM; pH 6.8) for 30 min. Afterward, each peptide domains of lectins specific to cell surface carbohydrates as the (Pep2854, Pep2855, and Pep2856) was covalently Gal-Man-(PO4) type and on the analysis of the active sites immobilized on a gold sensor chip using a 50 μg mL−1 which were previously identified and reported on the Protein peptide solution in MES buffer (50 mM; pH 7.0) by incu- Data Bank (PDB). The finding conserved domains in sAP and bation overnight to ensure a high density of bioreceptor on PPG are able to predict the interaction between peptides and the sensor chip surface. Finally, ethanolamine (1 M; these biomolecules which will mostly take place in these re- pH 8.5) was added to deactivate the unreactive carboxylic gions increasing the probability that the designed peptides can group. The sensor chips were rinsed with Milli-Q water and recognize any sAP or PPG secreted by a broad range number dried with N2. of Leishmania species. Therefore, from the Clustal Omega Design and characterization of high-affinity synthetic peptides as bioreceptors for diagnosis of cutaneous... results of the sAP secreted by Leishmania reported in UniProt, This finding gives excellent reliability and robustness to we selected the region corresponding to the molecular docking between these peptides and the gly- MVQVVHRHGARSALNDNTTEICGTLYPC (Code Job coproteins under study. Bo et al. show that the peptide se- UniProt: /www.uniprot.org/align/A20190117216DA2B77 quences of proteins often have intrinsic propensities for the BFBD2E6699CA9B6D1C41EB202DE21L), and for PPG, formation of their native conformations [30] which is incon- we chose the region corresponding to 1030–1450 of the sistent with our designed peptide that keeps the motifs struc- proteophosphoglycan with A4HM87 as the more conservative tural from lectins. region by the only four sequences reported (Code Job Uniprot: The most relevant physicochemical properties (i.e. mo- www.uniprot.org/align/A20190918674 lecular weight, net charge, isoelectric point, etc.) of the 6803381A1F0E0DB47453E0216320D05A710T.aln). The peptidomimetics are presented in Table 1. As for the mo- PDB codes, Clustal Omega, and the BLAST results are lecular dynamics performed, the CABS-dock server-gener- included in Fig. S1a–b of the ESM. ated ten top-ranked model structures of both the sAP- The peptides were synthesized and purified using Sepack peptide and the PPG-peptide complex. The docking poses C-18 columns, and the secondary structure was analysed by predicts the binding orientations of a ligand to the receptor circular dichroism as shown in Fig. 1. The structure of a lectin active site, many times defined by the user to evaluate sta- is reported in the Protein Data Bank with PDB code 2DHI. bilizing interactions such as hydrogen bonds, salt bridges, This protein belonging to this family is characterized by show- and hydrophobic contacts possible in the pairs of molecules ing mostly both beta-folded and α-helix motifs in theirs struc- using PDB files [31]. The computational experiment tures. In addition, the peptides retain the conformation, which allowed the full flexibility of the novel synthesized pep- is a very important observation considering that peptides hav- tides and a small fluctuation of the receptor backbone to ing less than 20 amino acids are crucial in the active design of explore the binding position according to the score function functional peptides that can access to a specific biological energies by calculating the root mean square deviation target. The Pep2854 and Pep2855 exhibited a strong tendency (RMSD) of each pose [32]. to a α-helix structure because of the presence of amino acids Three different RMSD classifications for docking solutions such as His, Cys, Glu, Asp, and Ala, while Pep2856 showed a were used as a reference: (i) right solution when RMSD ≤ ß-sheet profile, favoured by the presence of Gly, Val, Ile, and 2.0 Å; (ii) acceptable solution when RMSD is between 2.0 Leu residues (Fig. 2b–c). Our experimental data support the and 4.0 Å; and (iii) wrong solution when RMSD ≥ 4.0 Å results of the theoretical folding of the most likely conforma- [33]. The present work was oriented by prior knowledge tion peptides performed on the PEPFOLD server. [34, 35] of the interactions that may take place between three Fig. 1 Circular dichroism spectra a b 2,0x105 of the tendency of secondary Pep2854 structures of each synthetic 0,0 peptide in 30% v/v in Elip. Molar -2,0x105 trifluoroethanol with a peptide concentration of 0.1 mg mL−1. a -4,0x105 Secondary structure of a lectin reported with PDB code: 2DHI. b -6,0x105 Pep2854 2D-folding with an α- -8,0x105 helix structure tendency. c Pep2855 2D folding with an α- -1,0x106 helix structure stronger tendency. 190 195 200 205 210 215 220 d Pep2856 2D folding with a Wavelength(nm) beta-sheet tendency c 2,0x105 d 2,5x106 Pep2855 2,0x106 Pep2856 0,0 6 1,5x10 Elip. Molar Elip. Molar -2,0x105 1,0x106 5,0x105 -4,0x105 0,0 -5,0x105 -6,0x105 -1,0x106 -8,0x105 -1,5x106 -2,0x106 190 195 200 205 210 215 220 190 200 210 220 230 240 Wavelength (nm) Wavelength (nm) Prada Y.A. et al. Fig. 2 Top-ranked models obtained from CABS-dock docking with the conservative region (UniProt code: A4HM871030–1450) (d–f). The target peptide-RMSD values. The structures represent each of the peptides bound region of glycoprotein is coloured in green, and the peptide is coloured in close to a sAP fragment of the conservative region corresponding to blue. The dotted region represents the most likely site for binding MVQVVHRHGARSALNDNTTEICGTLYPC (a–c). The structures interaction according to the contact map represent each of the peptides bound close to the PPG binding at the peptides homologous to lectin and two different For Pep2855-PPG molecular dynamics, the smallest calculat- glycoconjugates. As shown in Fig. 2, the partial loss of the ed RMSD value was 3.48 Å; this value is on the borderline of local structure conformation of the peptides can be seen when a bad result. However, this must be considered since the cal- they are approaching the sAP region. The more frequent con- culation of RMSD increased significantly with the molecular tacts were between sAP residues Arg11, Ser 12, and Asn17, size of the MD structures; then, the RMSD became a data with and Pep2854 residues Asn7, Ser11, and Asn11 at a high- many attributes when the high dimensionality causes the al- frequency level (0.06–0.1) according to the contact maps. gorithms based on the clustering methods to calculate the For the dynamic molecular trajectories, the smallest RMSD trajectory poses to fail [36]. Hence, these data are considered of atomic coordinate value was 1.79 Å in the docking for as good approximations, which were complemented and cor- trajectories of the Pep2854-sAP pair, thus validating the related with the affinity counters obtained by subsequent SPR structure-based design process. We focused on the clustering experiments. based on the fewer RMSD values released to the peptide bond in the proximity of the binding site on the bioreceptor. For Determination of binding affinities by surface PPG in Fig. 2, it was hypothesised that Ser275, Ser289, and plasmon resonance biosensor analysis Ser363 of the proteophosphoglycan domain are the key bind- ing sites to the cationic peptides through Lys, Arg, and Glu The strategy used was the formation of the covalent amine residues, since in this case most interactions were salt bridges. coupling between the N-hydroxy succinimide (NHS- Table 1 Physicochemical properties of synthetic peptides ID peptide Sequence MW (Da) C* pI 2D folding #AA designed from lectin mannose- galactose-binding proteins Pep2854 HYSRTENMGTSNAC 1569.68 +1 8.58 Alpha-helix 14 Pep2855 KTFLKICKRPYVPSEP 1905.31 +4 10.5 Alpha-helix 16 Pep2856 INKQGDVTVLSNGNTQLT 1901.08 +1 10.14 Beta-sheet 18 *Net charge at pH 7.0; isoelectric point (pI); number of amino acidic residues (AA) Design and characterization of high-affinity synthetic peptides as bioreceptors for diagnosis of cutaneous... Fig. 3 Biofunctionalization strategy onto a plasmonic sensor chip for SPR analysis of the binding interaction between each of the synthetic peptides with both sAP and PPG activated) of a COOH-functional alkanethiol self-assembled kinetic parameters and the statistical parameter for all fitted monolayer (SAM) and the primary amine group present in curves (chi and R-square) 12. each peptide chain [37] (see Fig. 3). To facilitate the homoge- The sensorgrams resulting from the injection of the sAP neous distribution of the peptide monolayer on the surface and and PPG solution over the SAM without immobilized peptide minimize steric hindrance issues, hydroxyl-functional showed the full recovery of the baseline after the injection of alkanethiols were used as a lateral spacer in the SAM. the targets, confirming the absence of nonspecific adsorptions We independently evaluated the interaction of sAP and to the sensor surface at the best conditions of peptide immo- PPG with the three peptidomimetics, previously immobilized bilization (ESM Fig. S2). From the fitting equations in on the sensor chip. Results shown in Fig. 4 correspond to the Tables 2 and 3, it is possible to extract both kinetics (kon and fitting of the experimental sensorgram to the theoretical koff) and affinity parameters for the interaction. In terms of Prada Y.A. et al. Fig. 4 SPR analysis of peptide interactions with both sAP and PPG. The fitting curves are shown in dotted line. The figure included a sAP was tested in the concentration range from 5.2 to 104 nM, while for sensorgram of the interaction of sAP and PPG with the sensor surface PPG, the concentration range was from 3.0 to 58 nM, enough to cover the without immobilized peptide kinetic curves. The sensorgrams are shown in continuous lines, while Design and characterization of high-affinity synthetic peptides as bioreceptors for diagnosis of cutaneous... Table 2 Kinetic parameters of the interaction of synthetic peptides ID peptide KD (nM)±SE kon (×106 M s−1)±SE koff (s−1)±SE R-square (R2 ≈1) Chi (chi ≈ 0) with sAP Pep2854 1.48 ± 0.10 3.15 ± 0.23 0.004 ± 0.001 0.995 0.012 Pep2855 4.42 ± 0.09 4.49 ± 0.63 0.019 ± 0.002 0.997 0.025 Pep2856 15.6 ± 0.49 1.26 ± 0.30 0.020 ± 0.003 0.995 0.044 *SE, standard error of the mean. KD was estimated based on both steady-state affinity and kinetic steady-state 1:1 interaction model from three independent experiments kinetics, the association constant (kon) indicates the rapidness by other research where many peptide-based protein ligands, of the biomolecular interaction but, importantly, the dissocia- called synbodies, have high affinity and with a KD < 5 nM [39, tion constant (koff) is a measure of the stability of the complex. 40]. Nevertheless, synthetic peptides show some advantages In this sense, the Pep2854-sAP and Pep2856-PPG complexes over antibodies, such as their simple and robust production at are the most stable ones and their koff dissociation constants large scales and their high biochemical stability, therefore of- showed small values of 0.004 and 0.001 s−1, respectively. fering an interesting alternative for their use in diagnostic tests This observation can be interpreted as the fraction of the com- and devices. Finally, we demonstrated the specificity of the plex that dissociates every second, which is related to a longer peptides towards the molecules tested. The interaction by the half-life of these two molecular complexes. Regarding the kon peptides is not significant since an increase in the signal is measurement, we observed that Pep2855 quickly bounds to observed due to changes in the refractive index of the medium the two target molecules, showing similar kon values for both due to the bulky effect of BSA when it passes through the sAP (4.49 × 10−6 M s−1) and PPG (4.4 × 10−6 M s−1); howev- sensor surface (see ESM Fig. S5). er, these interactions were not the most stable. We infer from this finding that both Pep2854 and Pep2856 not only have a high affinity for the studied glycoproteins but also exhibit a distinct specificity for one of the targets. Affinity is the bind- Conclusions ing strength of a molecule to its ligand, and even though bind- ing is a reversible process, the measurement of the reaction We have designed and synthesized three high-affinity peptides rate constants can be used to define an equilibrium or affinity based on the galactolectin family, which can recognize constant (1/KD), so the smaller the KD value, the higher the Leishmania sAP and PPG proteins, considered as potential tar- affinity of the peptide for its target [37]. gets for the diagnosis of the infection. The dynamic molecular All peptides showed KD values in the nanomolar range, strategy employed allowed to model the most likely interactions evidencing the high affinity for sAP and PPG. The molecular between peptides and the targets selected, finding RMSD pairs, Pep2854-sAP and Pep2856-PPG, registered the values within the acceptable limits for theoretical calculations smallest KD values equal to 1.48 nM and 1.76 nM, respective- ( 3.0 Å). Pep2854 and Pep2856 exhibited the highest affinity ly, as shown in Tables 2 and 3. One of the most significant and specificity for sAP (KD of 1.48 nM) and PPG (KD of observation is that the interaction of the Pep2856-PPG was 9- 1.76 nM). These results support the hypothesis that peptides fold stronger than Pep2856-sAP, presumably because the pos- based on a rational design with the ability to occupy the binding itive charge of Pep2856 (+4) increases the electrostatic inter- site of a target glycoprotein can be used as high-affinity action with the negative groups in the PPG molecule at sites bioreceptors, being an alternative to large biomolecules as an- different to the active site. These affinity values are like the KD tibodies with limitations in terms of production, biochemical values of high-affinity antibodies, generally considered to be stability, and robustness. These peptides can be employed in in the range of 1 to 10−3 nM [38]. Our results demonstrated the development of new diagnostic tools for cutaneous leish- than the affinity values are similar to the KD values reported maniasis, especially for the construction of point-of-care Table 3 Kinetic parameters of the interaction of synthetic peptides ID peptide KD (nM)±SE kon (×106 M s−1)±SE koff (s−1)±SE R-square (R2 ≈1) Chi (chi ≈ 0) with PPG Pep2854 5.0 ± 0.26 2.6 ± 0.10 0.014 ± 0.005 0.995 0.014 Pep2855 2.7 ± 0.11 4.4 ± 0.09 0.012 ± 0.003 0.993 0.008 Pep2856 1.76 ± 0.50 1.8 ± 0.01 0.001 ± 0.002 0.987 0.019 *SE, standard error of the mean. KD was estimated based on both steady-state affinity and kinetic steady-state 1:1 interaction model from three independent experiments Prada Y.A. et al. biosensor devices that enable rapid and accurate detection of the 11. Ashrafmansouri MA, Sarkari B, Hatam G, Habibi P, Abdolahi Khabisi S. Utility of western blot analysis for the diagnosis of infection in decentralized and low-resource settings. cutaneous leishmaniasis. Iran J Parasitol. 2015;10(4):599–604. 12. de Paiva-Cavalcanti M, de Morais RCS, Pessoa-e-Silva R, et al. Leishmaniases diagnosis: an update on the use of immunological Supplementary Information The online version contains supplementary and molecular tools. Cell Biosci. 2015;5(1):1–10. https://doi.org/ material available at https://doi.org/10.1007/s00216-021-03424-2. 10.1186/s13578-015-0021-2. 13. Romero GAS, Orge MDLGO, Guerra MVDF, Paes MG, Macêdo Acknowledgements We thank Patricia Escobar from CINTROP for VDO, De Carvalho EM. Antibody response in patients with cuta- helpful discussion about Leishmania parasite. neous leishmaniasis infected by Leishmania (Viannia) braziliensis or Leishmania (Viannia) guyanensis in Brazil. Acta Trop. Funding This study was supported by a Doctoral Fellowship from 2005;93(1):49–56. https://doi.org/10.1016/j.actatropica.2004.09. COLCIENCIAS in Colombia. The ICN2 is funded by the CERCA 005. programme/Generalitat de Catalunya. ICN2 is supported by the Severo 14. Soulat D, Bogdan C. Function of macrophage and parasite phos- Ochoa program from Spanish Ministry of Science MICINN (Grant No. phatases in leishmaniasis. Front Immunol. 2017;8(DEC):1–21. SEV-2017-0706). This work has made use of the Biodeposition and https://doi.org/10.3389/fimmu.2017.01838. Biodetection Unit from ICTS NANBIOSIS (http://www.nanbiosis.es/ 15. Reiter-Owona I, Rehkaemper-Schaefer C, Arriens S, Rosenstock P, portfolio/u4-biodeposition-andbiodetection-unit/) partially supported by Pfarr K, Hoerauf A. Specific K39 antibody response and its persis- MICINN/FEDER (FICTS-1420-27). tence after treatment in patients with imported leishmaniasis. Parasitol Res. 2016;115(2):761–9. https://doi.org/10.1007/s00436- 015-4801-8. Declarations 16. Herrera G, Castillo A, Ayala MS, Flórez C, Cantillo-Barraza O, Ramirez JD. 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Odorranalectin is a Publisher’s note Springer Nature remains neutral with regard to jurisdic- small peptide lectin with potential for drug delivery and targeting. tional claims in published maps and institutional affiliations.