This is the accepted version of the journal article: Prada, Y. A.; Soler Aznar, Maria; Guzmán, Fanny; [et al.]. «Design and characterization of high-affinity synthetic peptides as bioreceptors for diagnosis of cutaneous leishmaniasis». Analytical and bioanalytical chemistry, Vol. 413, issue 17 (July 2021), p. 4545-4555. DOI 10.1007/s00216-021-03424-2 This version is available at https://ddd.uab.cat/record/248668 under the terms of the license 1 1 Design and characterization of high-affinity synthetic peptides as bioreceptors for 2 diagnosis of Cutaneous Leishmaniasis 3 4 Y.Andrea Prada,1 Maria Soler2, 3, Fanny Guzmán4, John J. Castillo5*, Laura M. Lechuga3,2 and Enrique 5 Mejía-Ospino1 6 1 Laboratorio de Espectroscopia Atómica y Molecular (LEAM), Universidad Industrial de Santander. Carrera 7 27 Calle 9, Bucaramanga, Santander, 678, Colombia. 8 2 Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Spain. 9 3 Nanobiosensors and Bioanalytical Applications Group (NanoB2A), Catalan Institute of Nanoscience and 10 Nanotechnology (ICN2), CSIC, and BIST, Campus UAB, Bellaterra, 08193, Barcelona, Spain. 11 4 Laboratorio de Síntesis de Péptidos, Núcleo de Biotecnología Curauma (NBC), Pontificia Universidad 12 Católica de Valparaíso, Valparaíso 2373223, Chile 13 5 Grupo de Investigación en Bioquímica y Microbiología (GIBIM), Universidad Industrial de Santander, 14 Bucaramanga 680002, Colombia. 15 16 Corresponding autor 17 +Autors email: *jcasleon@uis.edu.co 18 19 2 20 Graphical Abstract 21 22 Abstract 23 Cutaneous leishmaniasis (CL) is one of the illness caused by Leishmania parasites infection, which can be 24 asymptomatic or severe according to the Leishmania strain infecting. CL is commonly diagnosed by directly 25 detecting the parasites or their DNA in tissue samples. New diagnostic methodologies target specific proteins 26 (biomarkers) secreted by the parasite during its infection process. However, specific bioreceptors for the in vivo 27 or in vitro detection of these novel biomarkers are rather limited in terms of sensitivity and specificity. For this 28 reason, we here introduce three novel peptides as bioreceptors for the highly sensitive and selective 29 identification of acid phosphatase (sAP) and proteophosphoglycan (PPG), which have a crucial role in 30 leishmaniasis infection. These high-affinity peptides have been designed from the conservative domains of the 31 lectins family, holding the ability to interact with the biological target and produce the same effect that the 32 original protein. The synthetic peptides have been characterized and the affinity and kinetic constants for their 33 interaction with the targets (sAP and PPG) have been determined by a Surface Plasmon Resonance biosensor. 34 Values obtained for KD are in the nanomolar range, which is comparable to high-affinity antibodies, with the 35 additional advantage of a high biochemical stability and simpler production. Pep2854 exhibited a higher affinity 36 for sAP (KD =1.47 nM) while Pep2856 had a good affinity for PPG (K D 1.76 nM). This study evidences that 37 these peptidomimetics represent a novel alternative tool to the use of high molecular weight proteins for 38 biorecognition in the diagnostic test and biosensor devices for CL. 39 40 Keywords: Cutaneous leishmaniasis; high affinity peptides; lectins; protephosphoglycans; acid phosphatase 41 3 42 Introduction 43 Leishmaniasis, caused by Leishmania parasites, is one of the infectious diseases with the highest incidence in 44 the tropical regions of South America, Africa, the Mediterranean region, and Central Asia, affecting nearly 100 45 countries. According to the World Health Organization (WHO), there are one million cases of infection each 46 year, including asymptomatic cases which are not reported 1,2 . Leishmaniasis affects the most vulnerable 47 population in the world, usually associated with inadequate nutrition, immunosuppression, and deficiency in 48 the public health system. There are three clinical manifestations of leishmaniasis infection: cutaneous 49 leishmaniasis (CL), mucocutaneous leishmaniasis (ML), and visceral leishmaniasis (VL), known as kala-azar 50 3 . CL is the most frequent form of the disease; it causes skin lesions that fortunately can heal without treatment 51 in many cases 4. ML occurs when the parasite spreads into the nose, mouth, and throat, and it produces severe 52 ulcers, destroying the mucous membranes. It can be cured, but the symptoms might not be detected until years 53 after infection and, if not treated, it can evolve in serious complications 5. VL is the most lethal form of the 54 disease; it damages internal organs like spleen or liver, and it affects the bone marrow and the immune system, 55 which results fatal for the patient and death may occur due to complications associated with secondary 56 infections 6. 57 To date, there is no vaccine available against leishmaniasis. However, a recent study predicted 3D structure of 58 a vaccine candidate against VL caused by Leishamania donovani based on VC1/VC2 modelled using a 59 molecular dynamics coupled TLR-2 and TLR4 receptors 7. Therefore an accurate and early diagnosis of the 60 disease is crucial for the timely administration of treatment and the reduction of the infectious burden 8,9 . 61 Importantly, due to the geographic and sociologic distribution of leishmaniasis incidence, it is essential to 62 develop new diagnostic tests that can be implemented as point of care (POC) devices and applied for routine 63 screening analysis 10,11 . The broad spectrum of clinical manifestations of CL makes its timely diagnosis 64 complicated. Currently, standard methods for the diagnosis of CL include microscopy and microbiological 65 culture of the parasite, which require centralized laboratories, highly specialized personnel, and up to several 66 days for providing conclusive results 12 . The detection of CL via blood serological tests shows a very low 67 sensitivity due to a deficient humoral response by the immune system 13 . This is because the immunological 68 effect in humans is induced by the parasite amastigote form (reproductive phase) and not by the promastigote 69 (infective phase), which infects the macrophage cells immediately after contagion 14. Clinical assays targeting 70 gene sequences of the parasite using the polymerase chain reaction (PCR) technique have also been developed, 71 as well as immunochemical tests targeting antigens of the cell membrane, such as Kalazar Detect and IT-LEISH 72 for VL, based on recombinant Leishmania protein K39, and Montenegro skin test (MTS) for CL 15,16 . Other 73 candidates for the detection of leishmaniasis are the membrane glycoproteins gp63, gp70 and gp72 which are 74 specific for the Leishmania genus, and have improved the sensitivity and specificity of ELISA tests 17. 75 However, these are prone to cross-reactions with other diseases caused by trypanosomes family members. The 76 limitations of the conventional diagnostic methods are driving research efforts towards the finding of direct 77 biomarkers and potential targets to increase the sensitivity of the diagnosis 18. Recently, secreted proteins are 78 considered an untapped source of possible antigens and are being exploited using combinations of bioinformatic 4 79 and immunoproteomic approaches 19,20. Our focus is directed to the glycoproteins secreted during the infectious 80 process by Leishmania parasite, which are promising biomarkers for the detection of CL. Among them, the acid 81 phosphatases (sAP) and protephosphoglycans (PPG) are secreted by the parasite mostly during the infective 82 phase associated to the promastigote form; therefore, they can play a critical role as clinical targets for both 83 diagnosis and vaccine development 21. 84 The sAP are complex enzymes, N-carbohydrate and predominant O-linked phosphorylated, with molecular 85 weight between 90-130 kDa, that are considered a virulence factor 22 . The sAP inhibits the production of 86 superoxide anions by neutrophils and macrophages, closely related to the survival of the parasite inside the host 87 cell. The protephosphoglycans (PPG), with a molecular weight around 200 kDa, are involved in the mammalian 88 infection and may contribute to the binding of Leishmania to the host cell 23. 89 Nowadays, it is well known that the interaction of mannose-binding lectin with the specific carbohydrates of 90 glycoconjugates on the cell surface of Leishmania acts as an alternative pathway in the complement activation 91 mechanism antibody-independent response 24,25 . In this sense, lectins can recognize specific glycan 92 arrangements in the PPG molecules 26. The aim of this work is to design, synthesize, and characterize a new 93 peptidomimetic-based galactolectins with enhanced affinities and robustness for both sAP and PPG, to be used 94 for the highly sensitive detection of Leishmaniasis. [28,29]. 95 96 Based on the rational bioinformatic design and theoretical modelling of the interaction, we have obtained three 97 peptide sequences specifically targeting small regions (10-15 amino acid residues) of the lectin domains that 98 can keep the structure-activity relationship 27 . Peptides were synthesized using Fmoc solid-phase peptide 99 chemistry. Synthetic peptides have been fully characterized to determine the binding affinities and kinetic 100 parameters for the interaction with Leishmania PPG and sAP proteins by Surface Plasmon Resonance (SPR) 101 biosensor analysis 28 . Our findings demonstrate the valuable potential of the new synthetic peptides as 102 bioreceptors for the direct assay of leishmaniasis biomarkers in diagnostic tests. The higher affinity and stability 103 of our receptors might greatly enhance the analytical performance of conventional clinical assays or innovative 104 diagnostic devices, such as point-of-care biosensors. 105 106 Materials and Methods 107 General Chemistry 108 All common reagents and materials were obtained from a commercial source as Sigma Aldrich and MERCK 109 and used without further purification unless otherwise indicated. Glassware was oven-dried before use and the 110 solutions prepared in deionized water (Resistivity 18.2 MΩ). 111 Bioinformatic peptide design 5 112 The protein sequences of secreted acid phosphatase and proteophosphoglycans reported by Leishmania ssp 113 parasites were selected on Uniprot Data Bank (Available on: www.uniprot.org/). About twelve sequences of 114 sAP and four sequences of PPG with up to 60% identity analysed and aligned using the both, BLAST and 115 Clustal Omega tools (Available on Uniprot) for finding a conservative domain in an independent bioinformatic 116 analysis are reported in the Supplementary Information Figure. S1a-S1b. Subsequently, the native models for 117 both sAP and PPG conservative regions were obtained from the GalaxyWEB web server, which allowed making 118 the prediction, refinement, and optimization of the protein structure based on protein-template by rescoring 119 HHsearch results. Then a group of 15 sequences of glycoconjugate-recognized lectins were selected from 120 Protein Data Bank and ten fragment peptides between 14-18 amino acid residues of different motif regions of 121 the proteins which were extracted from Protein Data Bank (https://www.rcsb.org/) with ID codes: 1CIW; ICQ9; 122 ICR7; IQF3; 1RIR; 2DHI; 2DV9; 2DVA;2DVB;2DVD; 2DVF; 2DVG; 2DVP; 2TEP; 1BZW. The 2D 123 conformation of each peptide was modelled and visualized using the PEPFOLD server and PyMol v2.3 124 respectively. The experimental data suggest that three peptides sequences described as Pep2854, Pep2855, and 125 Pep2856 adopted a conformational space-optimized based on high interaction similarity with the targets. 126 Theoretical modelling of peptide-glycoproteins interactions 127 CABS-Dock server (http://biocomp.chem.uw.edu.pl/CABSdock) was used to investigate the protein-peptide 128 interaction. For the performance of the dynamic simulation, the algorithms found the binding site with full 129 spatial flexibility of peptide and decreased the fluctuation on the protein backbone without an a priori 130 knowledge of the binding site 29. To evaluate the results, the RMSD value was used, which is defined as the 131 root mean square deviation of the peptide-protein interaction. For selecting the better models, the contact maps 132 were calculated with a cut-off distance value of 4.5 Å for each peptide-glycoconjugate complex (Supporting 133 Information Figure. S3a-S3b). 134 Synthesis and purification of peptides 135 Pep2854 (HYSRTENMGTSNAC), Pep2855 (KTFLKICKRPYVPSEP) and Pep2856 136 (INKQGDVTVLSNGNTQLT) were synthesized by solid-phase peptide synthesis following the Fmoc 137 methodology. Briefly, peptides building blocks were loaded on of Rink amide 4MBHA resin (0.55 mmol/g 138 substitution;100 mg per tea-bag) followed by chain linkage via the coupling of Fmoc-protected-L-amino acids 139 between the C-terminal residue and the resin. The amino acids were coupled using a mixture of the L-amino 140 acid, O-(Benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) or , oxime and N- 141 Ethyldiisopropylamine (DIEA) in this ration 5:5:5:7.5 respectively for over 3 hours. The Fmoc protecting 142 groups were removed with piperidine (20% v/v in 0.01% Tween 20 solution). All coupling reactions were 143 carried out in dimethylformamide. The peptides were cleaved and the lateral chain protecting groups were 144 removed using a cocktail containing trifluoroacetic acid (TFA), triisopropylsilane (TIS) and water (ratio 145 95:2.5:2.5). Lastly, the peptides were precipitated with cold diethyl ether, centrifuged thrice and the pellet was 146 dissolved in water before lyophilization. The crude peptides were purified by RP-HPLC on a Zorbax C-18 6 147 preparative column (7.0 µm; 21.5 x 250 mm) using a gradient from 30% to 70% of acetonitrile (ACN) (Mobil 148 phase A: H2O, 0.001% TFA and B: ACN, 0.001% TFA. 149 Mass spectrometry analysis 150 The fractions collected from RP-HPLC were analyzed by MALDI-TOF spectrometry in an UltrafleXtreme 151 Bruker Daltonics mass spectrometer using a dried-droplet method using an α-Cyano-4-hydroxycinnamic acid 152 saturated solution (0.001% TFA in ACN/ H2O) as matrix substance for MALDI. 153 154 Circular dichroism analysis 155 2D peptide structures were analyzed by circular dichroism in a Jasco J-815 spectropolarimeter with an 156 acquisition speed of 20 nm/min in a spectral region of 190-260 nm. 157 Surface Plasmon Resonance biosensor 158 SPR experiments were performed at room temperature (25°C) on a home-made SPR biosensor. The sensor 159 platform is based on the Kretschmann configuration that allowed the monitoring of the spectrum of the reflected 160 light at 70° of incident angle on a 50 nm layer of the gold sensor chip, previously biofunctionalized with each 161 peptide. The biosensor surface is clamped in a flow cell and is irradiated with a halogen lamp (HL-2000, Ocean 162 Optics USA) in transverse-electric polarization mode; the light passes through a trapezoidal prism for plasmonic 163 excitation. Then, the reflected light is collected by an optical fiber-coupled to a CCD spectrometer (Ocean 164 Optics-Jazz Module, USA). The microfluid delivery system includes a syringe pump (New Era, NE1000, USA), 165 connected to the flow cell. Samples of sAP and PPG were manually injected using an injection valve (IDEZ 166 Health and Science, V-451, USA). Thereby, the biomolecular interactions that take place on the sensor surface 167 can be detected by the changes in the refraction index and the displacement of the plasmon wavelength (ΔλSPR). 168 Data are presented as a sensorgram where the ΔλSPR is evaluated in real-time. 169 170 Sensor surface biofunctionalization 171 The sensor chip is based on an ultra-thin gold layer (50 nm thickness) deposited on a glass slide previously 172 covered with titanium (2.0 nm thickness). The sensor chips were washed with acetone, isopropanol, and Milli- 173 Q water by immersing at boiling temperature. After that, they were dried with N 2 and placed in an ozone cleaner 174 (BioForce Nanoscience, USA) for 40 min. A self-assembled monolayer (SAM) was carried out ex-situ using a 175 mixture of functional alkanethiols, mercaptohexadecanoic acid (MHDA) and mercaptoundecanol (MUOH) in 176 a 1:1 ratio and total concentration of 250 µM in ethanol, where the sensor chips were incubated overnight. Then, 177 the nanoplasmonic sensors were rinsed with ethanol and dried with N 2. The carboxylic group activation was 178 performed via carbodiimide esters formation using a mixture of 1-etil-3-(3-dimethylaminopropil)carbodiimide 179 (EDC) and N-hydroxysuccinimide ester (NHS) (0.2 M/0.05 M) solution in 2-(N-morpholino)ethanesulfonic 7 180 acid (MES) buffer (100 mM; pH 6.8) for 30 min. Afterward, each peptide (Pep2854, Pep2855, and Pep2856) 181 was covalently immobilized on a gold sensor chip using a 50 µg mL-1 peptide solution in MES buffer (50 mM; 182 pH 7.0) by incubation overnight to ensure a high density of bioreceptor on the sensor chip surface. Finally, 183 ethanolamine (1M; pH 8.5) was added to deactivate the unreactive carboxylic group. The sensor chips were 184 rinsed with Milli-Q water and dried with N2. 185 186 Binding interaction analysis on sensor chips 187 The dilution series of targets were injected (sAP and PPG) in separate experiments for each peptide without 188 exceeding the saturation concentration of the analyte onto the sensor chip surface. In the case of sAP, the range 189 of concentrations injected was from 5.2 to 104 nM in 10 mM MES buffer pH 6.5. Moreover, the PPG was 190 injected in concentrations between 3.0 and 58 nM prepared in 10 mM acetate buffer pH 4.5. The running buffer 191 for each assay was the same as for the sample to avoid a bulk refractive index effect, and the flow rate was 30 192 µL/min applied for 15 min. The regeneration step was carried out so that all peptides were able to dissociate 193 entirely using 5.0 mM of NaOH at a flow rate of 30 µL/min for 2 min. The sensorgrams acquired were fitted 194 to a one-site binding interaction model using GraphPrism software (Version 9.0). This model assumed that the 195 formation of the target-ligand complex is not limited by mass transport because the SPR biosensor is capable 196 of keeping a constant target concentration in a continuous flow. Therefore, in the kinetic analysis, the constants 197 of association (kon) and dissociation (koff) were calculated from the plot of ΔλSPR versus concentration30. 198 Consequently, the equilibrium dissociation constant (KD), known as affinity, was determined from the relation 199 KD= koff/kon. Additionally, the Chi2 value was reported as a parameter indicative of the fitting confidence. 200 For the determination of SPR sensitivity we used buffer PBS serial solutions prepared in MilliQ water; then 201 refraction index of each PBS solution was measured in a Refractometer and were injected in order of less to 202 major concentration. Then Δλ was determined from the LSPR vs Real time graphical as shown in the Figure. 203 S4 of Supporting Information. 204 205 Results and analysis 206 Design and synthesis of specific PPG and sAP bioreceptor peptides 207 We have developed three new peptidomimetic molecules for the sensitive and selective detection of PPC and 208 sAP proteins, secreted by Leishmania parasite during the infection process. The rational design of peptides was 209 based on the conservative domains of lectins specific to cell-surface carbohydrates as the Gal-Man-(PO4) type 210 and on the analysis of the active sites which were previously identified and reported on the Protein Data Bank 211 (PDB). The finding conserved domains in sAP and PPG are able to predict the interaction between peptides and 212 these biomolecules which will mostly take place in these regions increasing the probability that the peptides 213 designed can recognize any sAP or PPG secreted by a broad range number of Leishmania species. Therefore, 214 from the Clustal Omega results of the sAP secreted by Leishmania reported in Uniprot, we selected the region 8 215 corresponding to MVQVVHRHGARSALNDNTTEICGTLYPC (Code Job Uniprot: 216 /www.uniprot.org/align/A20190117216DA2B77BFBD2E6699CA9B6D1C41EB202DE21L) and for PPG we 217 choose the region corresponding to 1030-1450 of the proteophosphoglycan with A4HM87 as more conservative 218 region by the only four sequences reported (Code Job 219 Uniprot:www.uniprot.org/align/A201909186746803381A1F0E0DB47453E0216320D05A710T.aln). The 220 PDB codes, Clustal Omega and the BLAST results are included in the Supporting Information in the Figure. 221 S1a-S1b. 222 The peptides were synthesized and purified using Sepack C-18 columns, and the 2D structure was analysed by 223 circular dichroism as shown in Fig. 1. The 2D structure of a lectin reported in the Protein Data Bank with PDB 224 code 2DHI. These proteins family are characterized by showing mostly both beta-folded and an α-helix motifs 225 structure. In addition, the peptides retain the 2D structure, a very important observation considering that 226 peptides having less than 20 amino acids are crucial in the active design of functional peptides that can access 227 to a specific biological target. The Pep2854 and Pep2855 exhibited a strong tendency to an α-helix structure as 228 a result of the presence of amino acids such as His, Cys, Glu, Asp and Ala, while Pep2856 showed ß-sheet 229 profile, favoured by the presence of Gly, Val, Ile, and Leu residues (Fig. 2b-2c). Our experimental data support 230 the results of the theoretical folding of the most likely conformation peptides performed on the PEPFOLD 231 server. a b 2,0x105 Pep2854 0,0 Elip. Molar 5 -2,0x10 -4,0x105 -6,0x105 -8,0x105 -1,0x106 190 195 200 205 210 215 220 Wavelength (nm) c d 2,0x105 Pep2855 2,5x106 0,0 2,0x106 Pep2856 1,5x106 Elip. Molar 5 Elip. Molar -2,0x10 1,0x106 5 5,0x105 -4,0x10 0,0 -6,0x105 -5,0x105 -1,0x106 -8,0x105 -1,5x106 190 195 200 205 210 215 220 -2,0x106 190 200 210 220 230 240 Wavelength (nm) Wavelength (nm) 232 233 Fig. 1. Circular dichroism spectra of the tendency of secondary structures of each synthetic peptide in 30% v/v 234 in trifluoroethanol with a peptide concentration of 0.1 mg mL-1. a) 2D structure of a lectine reported with PDB 235 code: 2DHI. b) Pep2854 2D-folding with an α-helix structure tendency. c) Pep2855 2D-folding with an α-helix 236 structure stronger tendency. d) Pep2856 2D-folding with a beta-sheet tendency. 237 9 238 This finding gives excellent reliability and robustness to the molecular docking between these peptides and the 239 glycoproteins under study. Bo et al. shows that the peptide sequences of proteins often have intrinsic 240 propensities for the formation of their native conformations 31 which is in consistent with our designed peptide 241 that keeps the motifs structural from Lectins. 242 The most relevant physicochemical properties (i.e., molecular weight, net charge, isoelectric point, etc.) of the 243 peptidomimetics are presented in Table 1. As for the molecular dynamics performed, the CABS-Dock server- 244 generated ten top-ranked model structures of both the sAP-peptide and the PPG-peptide complex. The docking 245 poses predict the binding orientations of a ligand to the receptor active site, many times defined by the user to 246 evaluate stabilizing interactions such as hydrogen bonds, salt-bridges, and hydrophobic contacts possible in the 247 pairs of molecules using PDB files 32. The computational experiment allowed the full flexibility of the novel 248 synthesized peptides and a small fluctuation of the receptor backbone to explore the binding position according 249 to the score function energies by calculating the root mean square deviation (RMSD) of each pose 33. 250 251 Table 1. Physicochemical properties of synthetic peptides designed from lectins mannose-galactose binding 252 proteins. ID 2D- Sequence MW (Da) C* pI folding #AA Peptide Alpha- Pep2854 HYSRTENMGTSNAC 1569.68 +1 8.58 14 helix Alpha- Pep2855 KTFLKICKRPYVPSEP 1905.31 +4 10.5 16 helix Betha- Pep2856 INKQGDVTVLSNGNTQLT 1901.08 +1 10.14 18 sheet 253 *Net charge at pH 7.0; isoelectric point (pI); Number of amino acidic residues (AA). 254 255 Three different RMSD classifications for docking solutions were used as a reference: i) right solution when 256 RMSD ≤ 2.0 Å; ii) acceptable solution when RMSD is between 2.0 and 4.0 Å; and iii) wrong solution when 257 RMSD ≥ 4.0 Å 34. The present work was oriented by prior knowledge 35,36 of the interactions that could take 258 place between three peptides homologous to lectin and two different glycoconjugates as shown in Fig. 2, the 259 partial loss of the 2D conformation of the peptides can be seen when they are approaching the sAP region. The 260 more frequent contacts were between sAP residues Arg11, Ser 12 and Asn17 and Pep2854 residues Asn7, 261 Ser11, Asn11 at a high-frequency level (0.06-0.1) according to the contact maps. 10 262 263 Fig. 2. Top-ranked models obtained from CABS-Dock docking with the peptide-RMSD values. The structures 264 represent each of the peptides bound close to a sAP fragment of the conservative region corresponding to 265 MVQVVHRHGARSALNDNTTEICGTLYPC (a-c). The structures represent each of the peptides bound close 266 to the PPG binding at the conservative region (Uniprot code: A4HM87 1030-1450) (d-f). The target region of 267 glycoprotein is coloured in green and the peptide is coloured in blue. The dotted region represents the most 268 likely site for binding interaction according to the contact map. 269 270 For the dynamic molecular trajectories, the smallest RMSD of atomic coordinates value was 1.79 Å in the 271 docking for trajectories of the Pep2854-sAP pair, thus validating the structure-based design process. We 272 focused on the clustering based on the fewer RMSD values released to the peptide bond in the proximity of the 273 binding site on the bioreceptor. For PPG in Fig. 2, it was hypothesized that Ser275, Ser289, and Ser363 of the 274 protephosphoglycans domain are the key binding sites to the cationic peptides through Lys, Arg and Glu 275 residues, for this case most interactions were salt bridges. For Pep2855-PPG molecular dynamics, the smallest 276 calculated RMSD value was 3.48 Å; this value is on the borderline of a bad result. However, this must be 277 considered since the calculation of RMSD increased significantly with the molecular size of the MD structures; 278 then, the RMSD became on data with many attributes when the high dimensionality causes that the algorithms 279 based on the clustering methods to calculate the trajectory poses to fail 37. Hence, these data are considered as 280 good approximations, which were complemented and correlated with the affinity counters obtained by 281 subsequent SPR experiments. 11 282 Determination of binding affinities by Surface Plasmon Resonance biosensor analysis 283 SPR biosensor technique plays a prominent role in biomolecular interactions characterization due to its ability 284 to detect and monitor molecular binding events in real time without the use of labels. A home-made SPR 285 biosensor working in Kretschmann configuration was employed for the analysis and determination of the 286 binding affinities and kinetic parameters of the interactions between the target proteins and the new peptides. 287 The working principle of the SPR biosensor is based on the high sensitivity of an evanescent plasmonic field 288 generated at the interface of a nanometric metal layer (i.e. 50 nm of gold) and the dielectric medium when the 289 light strikes at a specific angle. The properties of the evanescent field depend significantly on the refractive 290 index of the medium whereby the small changes due to molecular interactions can be measured, as well as the 291 formation of molecular pairs (target-bioreceptor) that takes place on the sensor surface. The SPR analysis 292 requires that one of the studied molecules is immobilized on the gold surface chip. The strategy used was the 293 formation of the covalent amine coupling between the N-Hydroxy succinimide (NHS-activated) of a COOH- 294 functional alkanethiol self-assembled monolayer (SAM) and the primary amine group present in each peptide 295 chain 38 (see Fig. 3). To facilitate the homogeneous distribution of the peptide monolayer on the surface and 296 minimize steric hindrance issues, hydroxyl-functional alkanethiols were used as a lateral spacer in the SAM. 297 Fig. 3. Biofunctionalization strategy onto a plasmonic sensor chip for SPR analysis of the binding interaction 298 between each of the synthetic peptides with both sAP and PPG. 12 299 We independently evaluated the interaction of sAP and PPG with the three peptidomimetics, previously 300 immobilized on the sensor chip. Results shown in Fig. 4 correspond to the fitting of the experimental 301 sensorgram to the theoretical kinetic parameters and the statistical parameter for all fitted curves (Chi and R- 302 square).12 303 Fig. 4. SPR analysis of peptidomimetics interactions with both sAP and PPG. The sAP was tested in the 304 concentration range from 5.2 to 104 nM, while for PPG, the concentration range was from 3.0 to 58 nM, enough 305 to cover the kinetic curves. The sensorgrams are shown in continuous lines, while fitting curves are shown in 306 dotted line. 307 The sensorgrams resulting from the injection of the sAP and PPG solution over the SAM without immobilized 308 peptide showed the full recovery of the baseline after the injection of the targets, confirming the absence of 309 nonspecific adsorptions to the sensor surface at the better conditions of peptide immobilization (Supporting 310 Information Figure. S2). In the Table 2-3 from the fitting equations, it is possible to extract both kinetics (k on 311 and koff) and affinity parameters for the interaction. In terms of kinetics, the association constant (k on) indicates 312 the rapidness of the biomolecular interaction but, importantly, the dissociation constant (k off) is a measure of 313 the stability of the complex. In this sense, the Pep2854-sAP and Pep2856-PPG complexes are the most stable 314 ones and their koff dissociation constant showed small values of 0.004 and 0.001 s-1, respectively. This 315 observation can be interpreted as the fraction of the complex that dissociates every second, which is related to 316 a longer half-life of these two molecular complexes. Regarding the kon measurement, we observed that Pep2855 317 quickly bounds to the two target molecules, showing similar kon values for both sAP (4.49x10-6 M s-1) and PPG 318 (4.4x10-6 M s-1), however these interactions were not the most stable. We infer from this finding that both 319 Pep2854 and Pep2856 not only have a high affinity for the studied glycoproteins but also exhibit a distinct 320 specificity for one of the targets. Affinity is the binding strength of a molecule to its ligand, though the binding 13 321 is a reversible process, the measurement of the reaction rate constants can be used to define an equilibrium or 322 affinity constant (1/KD), so the smaller the KD value, the higher the affinity of the peptide for its target [38]. 323 Table 2. Kinetic parameters of the interaction of synthetic peptides with sAP K (nM) k (x106 M-s-1) k (s-1) R-square Chi ID Peptide D on off (R2 ≈ 1) (Chi ≈ 0) Pep2854 1.47 3.15 0.004 0.995 0.012 Pep2855 4.42 4.49 0.019 0.997 0.025 Pep2856 15.6 1.26 0.020 0.995 0.044 324 325 326 Table 3. Kinetic parameters of the interaction of synthetic peptides with PPG K (nM) k (x106M-s-1) k (s-1) R-square Chi ID Peptide D on off (R2 ≈ 1) (Chi ≈ 0) Pep2854 5.0 2.6 0.014 0.995 0.014 Pep2855 2.7 4.4 0.012 0.993 0.008 Pep2856 1.76 1.8 0.001 0.987 0.019 327 328 All peptides showed KD values in the nanomolar range, evidencing the high affinity for sAP and PPG. The 329 molecular pairs, Pep2854-sAP and Pep2856-PPG, registered the smallest KD values equal to 1.47 nM and 1.76 330 nM, respectively, as shown in Table 2-3. One of the most significant observation is that the interaction of the 331 Pep2856-PPG was 9-fold stronger than Pep2856-sAP, presumably because the positive charge of Pep2856 (+4) 332 increases the electrostatic interaction with the negative groups in the PPG molecule at sites different to the 333 active site. These affinity values are like the KD values of high-affinity antibodies, generally considered to be 334 in the range of 1 to 10-3 nM 39. Nevertheless, synthetic peptides show some better properties than antibodies, 335 such as their simple and robust production at large scales and their high biochemical stability, therefore offering 336 an interesting alternative for their use in diagnostic tests and devices. Finally, we demonstrate the specificity of 337 the peptides towards the molecules tested. The interaction by the peptides is not significant since an increase in 338 the signal is observed due to changes in the refractive index of the medium due to the bulky effect of BSA when 339 it passes through the sensor surface (see Figure S5). 340 Conclusions 341 We have designed and synthesized three high-affinity peptidomimetics based on the galactolectins family, 342 which can recognize Leishmania sAP and PPG proteins, considered as potential targets for the diagnosis of the 343 infection. The dynamic molecular strategy employed allowed to model the most likely interactions between 344 peptides and the targets selected, finding RMSD values within the acceptable limits for theoretical calculations 14 345 (⁓3.0 Å). Pep2854 and Pep2856 exhibited the highest affinity and specificity for sAP (KD of 1.47 nM) and PPG 346 (KD of 1.76 nM). These results support the hypothesis that peptides based on a rational design with the ability 347 to occupy the binding site of a target glycoprotein can be used as high affinity bioreceptors, as an alternative to 348 large biomolecules as antibodies with limitations in terms of production, biochemical stability and robustness. 349 These peptides can be employed in the development of new diagnostic tools for cutaneous leishmaniasis, 350 especially for the construction of point-of-care biosensor devices that enable rapid and accurate detection of the 351 infection in decentralized and low-resource settings. 352 Conflicts of interest 353 There are no conflicts to declare. 354 Acknowledgment 355 We thank Patricia Escobar from CINTROP for helpful discussion about Leishmania parasite. This study was 356 supported by Doctoral Fellowship from COLCIENCIAS in Colombia. The ICN2 is funded by the CERCA 357 programme/Generalitat de Catalunya. 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