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Short catalytic peptides offer simple, tunable, low-cost biomolecules able to catalyze chemical reactions such as ester hydrolysis 1 . The main drawback of this approach is their low catalytic efficiency attributed to the lack of well-defined tridimensional structures characteristic for enzymes. Peptide self-assembly offers the possibility to obtain nanostructures with a higher degree of order and improved catalytic efficiency. However, better understanding of the principles that govern the catalytic activity of short peptides at the sequence level is needed. Our aim is to design short peptides, inspired by active sites found in hydrolases containing the catalytic triad. For this purpose, the amino acids sequences of 23 hydrolases acting on ester bonds (EC 3.1.) were shortened to peptides composed of the catalytic triad and the residues forming the oxyanion hole. We present the design of three sequences that will be evaluated experimentally: SGNYDYLHGD, CTLGLGSHCGG, GGESTGHTGAGNDK obtained from the active sites of 1-alkyl-2- acetylglycerophosphocholine esterase and protein- glutamate methylesterases (CheB and CheD), respectively. The sequences were designed to include the core fragment derived from the active site containing important residues found in proximity at the sequence level. In the case of 1- alkyl-2- acetylglycerophosphocholine esterase, the core sequence is based on the Tyr191- His195 segment. Other important residues that are far away from the selected core, are added at the N- or C- terminus, respecting their original sequence order (e.g. Ser47, Gly74 and Asn104 were added at the N- terminus of the YDYLH core sequence). Additionally, residues that allow the cyclization were included at the C- terminus, where Gly acts as spacer and Asp is used for the head-to-side- chain cyclization with the N- terminal amino acid. The peptides were synthesized by solid-phase peptide synthesis (SPPS) as linear and cyclic analogues. The cyclization was achieved by three different methods (head-to-side-chain, cysteine mediated and coupling of side chains) depending on the sequence composition. The peptides were purified using semipreparative-HPLC and analyzed using LC-MS and analytical HPLC. Linear and cyclic analogues of the proposed sequences will be tested to assess whether the enhanced rigidity obtained by cyclization will improve their catalytic activity. The peptides will be further analyzed using FTIR, CD, fluorescence spectroscopy (CAC) to investigate their ability to self-assemble and to determine how the cyclization affects nanostructure formation. The catalytic efficiency of ester hydrolysis for both linear and cyclic analogues will be evaluated and compared using the standard colorimetric test with 4-nitrophenyl acetate (p- NPA). A successful outcome of this project could result in general rules applicable to the design of short catalytic peptides with esterase activity.

Short catalytic peptides are tunable, low-cost biomolecules able to catalyze chemical reactions such as ester hydrolysis1. The main drawback is their low catalytic efficiency attributed to the lack of well-defined tridimensional structures characteristic for enzymes. Peptide self-assembly offers the possibility to obtain nanostructures with a higher degree of order leading to improved catalytic efficiency2. Alternatively, the cyclization of linear peptides restrains their conformational freedom, stabilizing the active site. Our aim is to design short catalytic linear and cyclic peptides to assess whether the enhanced rigidity obtained by cyclization improves their catalytic efficiency. The design of the sequences was inspired by active sites found in hydrolases acting on ester bonds (EC 3.1.) and it was based on the selection of a fragment containing the triad, from the primary sequence of the active site, retaining its chemical environment (Figure 1). Selected sequences from the 1-alkyl-2- acetylglycerophosphocholine esterase and protein- glutamate methylesterases (CheD and CheB) were synthesized using solid-phase peptide synthesis (SPPS) approach. The cyclization was achieved by three different methods (head-to-side chain, disulfide bridge formation and side chain-to-side chain) depending on the sequence composition. The peptides were purified using semipreparative-HPLC and characterized by LC-MS and analytical HPLC. The catalytic activities of the linear and cyclic peptides were evaluated using the 4-nitrophenyl acetate (p-NPA) colorimetric test and the obtained results will be described. Additionally, analogues of cysteine-containing peptides were designed and synthesized. The oxidation kinetics and the effect of thiol groups on catalytic activity were investigated. A successful outcome of this project will contribute to a better understanding of the relationship between short peptide sequences and their catalytic activity applicable to peptides with esterase activity.

Short catalytic peptides are tunable, low-cost biomolecules able to catalyze chemical reactions such as ester hydrolysis 1 . The main drawback is their low catalytic efficiency attributed to the lack of well-defined tridimensional structures characteristic for enzymes. Peptide self-assembly offers the possibility to obtain nanostructures with a higher degree of order leading to improved catalytic efficiency. Alternatively, the cyclization of linear peptides restrains their conformational freedom, stabilizing the active site. Our aim is to design short catalytic linear and cyclic peptides to assess whether the enhanced rigidity obtained by cyclization improves their catalytic efficiency. The design of the sequences was inspired by active sites found in hydrolases acting on ester bonds (EC 3.1.) and it was based on the selection of a fragment containing the triad, from the primary sequence of the active site, retaining its chemical environment (Figure 1). Three sequences (SGNYDYLHGE, CTLGLGSHCGG, and GGESTGHTGAGNDK) obtained from the 1-alkyl-2 acetylglycerophosphocholine esterase and protein- glutamate methylesterases (CheD and CheB), respectively, were synthesized using solid-phase peptide synthesis (SPPS) approach. The cyclization was achieved by three different methods (head-to- side chain, disulfide bridge formation and side chain-to-side chain) depending on the sequence composition. The peptides were purified using semipreparative-HPLC and characterized by LC-MS and analytical HPLC, while their ability to self- assemble and form possible nanostructures was assessed through FTIR, CD and fluorescence spectroscopy analyses. The catalytic activities of the linear and cyclic peptides were evaluated using the 4-nitrophenyl acetate (p-NPA) colorimetric test and the obtained results will be described. A successful outcome of this project will contribute to a better understanding of the relationship between short peptide sequences and their catalytic activity applicable to peptides with esterase activity.

Copyright © 2021 American Chemical Society, Passing through the blood-brain barrier (BBB) to treat neurological conditions is one of the main hurdles in modern medicine. Many drugs with promising in vitro profiles become ineffective in vivo due to BBB restrictive permeability. In particular, this includes drugs such as antiviral porphyrins, with the ability to fight brain-resident viruses causing diseases such as HIV-associated neurocognitive disorders (HAND). In the last two decades, BBB shuttles, particularly peptide-based ones, have shown promise in carrying various payloads across the BBB. Thus, peptide–drug conjugates (PDCs) formed by covalent attachment of a BBB peptide shuttle and an antiviral drug may become key therapeutic tools in treating neurological disorders of viral origin. In this study, we have used various approaches (guanidinium, phosphonium, and carbodiimide-based couplings) for on-resin synthesis of new peptide–porphyrin conjugates (PPCs) with BBB-crossing and potential antiviral activity. After careful fine-tuning of the synthetic chemistry, DIC/oxyma has emerged as a preferred method, by which 14 different PPCs have been made and satisfactorily characterized. The PPCs are prepared by coupling a porphyrin carboxyl group to an amino group (either N-terminal or a Lys side chain) of the peptide shuttle and show effective in vitro BBB translocation ability, low cytotoxicity toward mouse brain endothelial cells, and low hemolytic activity. Three of the PPCs, MP-P5, P4-MP, and P4-L-MP, effectively inhibiting HIV infectivity in vitro, stand out as most promising. Their efficacy against other brain-targeting viruses (Dengue, Zika, and SARS-CoV-2) is currently under evaluation, with preliminary results confirming that PPCs are a promising strategy to treat viral brain infections., Work supported by the La Caixa Health Foundation (project HR17_00409, ID 100010434, agreement LCF/PR/HR17/52150011), by the European Union (H2020-FETOPEN-2018-2019-2020-01 grant no 828774), and by the Spanish Ministry of Science and Innovation (AEI/FEDER grant CTQ2017-84371-P). NMR experiments were performed in the “Manuel Rico” NMR laboratory, LMR, CSIC, a node of the Spanish Large-Scale National Facility ICTS R-LRB. Additional funding from Fundação para a Ciência e Tecnologia (FCT-MCTES) is also acknowledged for DA Mendonça (PD/BD/136752/2018) and for C. Cruz-Oliveira and I. Cadima-Couto (PTDC/BIA-VIR/29495/2017).

Short catalytic peptides offer simple, tunable, low-cost biomolecules able to catalyze chemical reactions such as ester hydrolysis1. The main drawback of this approach is their low catalytic efficiency attributed to the lack of well-defined tridimensional structures characteristic for enzymes. Peptide self-assembly offers the possibility to obtain nanostructures with a higher degree of order and improved catalytic efficiency. However, better understanding of the principles that govern the catalytic activity of short peptides at the sequence level is needed. Our aim is to design short peptides, inspired by active sites found in hydrolases containing the catalytic triad. For this purpose, the amino acids sequences of 23 hydrolases acting on ester bonds (EC 3.1.) were shortened to peptides composed of the catalytic triad and the residues forming the oxyanion hole. We present the design of three sequences that will be evaluated experimentally: SGNYDYLHGD, CTLGLGSHCGG, GGESTGHTGAGNDK obtained from the active sites of 1-alkyl-2- acetylglycerophosphocholine esterase and protein- glutamate methylesterases (CheD and CheB), respectively. The sequences were designed to include the core fragment derived from the active site containing important residues found in proximity at the sequence level. In the case of 1- alkyl-2-acetylglycerophosphocholine esterase, the core sequence is based on the Tyr191-His195 segment. Other important residues that are far away from the selected core, are added at the N- or C-terminus, respecting their original sequence order (e.g. Ser47, Gly74 and Asn104 were added at the N-terminus of the YDYLH core sequence). Additionally, residues that allow the cyclization were included at the C-terminus, where Gly acts as spacer and Asp is used for the head-to-side-chain cyclization with the N-terminal amino acid. The peptides were synthesized by solid-phase peptide synthesis (SPPS) as linear and cyclic analogues. The cyclization was achieved by three different methods (head-to-side-chain, cysteine mediated and coupling of side chains) depending on the sequence composition. The peptides were purified using semipreparative-HPLC and analyzed using LC-MS and analytical HPLC. Linear and cyclic analogues of the proposed sequences will be tested to assess whether the enhanced rigidity obtained by cyclization will improve their catalytic activity. The peptides will be further analyzed using FTIR, CD, fluorescence spectroscopy (CAC) to investigate their ability to self-assemble and to determine how the cyclization affects nanostructure formation. The catalytic efficiency of ester hydrolysis for both linear and cyclic analogues will be evaluated and compared using the standard colorimetric test with 4-nitrophenyl acetate (p- NPA). A successful outcome of this project could result in general rules applicable to the design of short catalytic peptides with esterase activity.

Additional file 1. Supporting information for Algorithm-supported, mass and sequence diversity-oriented random peptide library design

Additional file 2. List of amino acid permutations for BS 2, from the optimization in Fig. 4.

Additional file 2. List of amino acid permutations for BS 2, from the optimization in Fig. 4.

Passing through the blood-brain barrier (BBB) to treat neurological conditions is one of the main hurdles in modern medicine. Many drugs with promising in vitro profiles become ineffective in vivo due to BBB restrictive permeability. In particular, this includes drugs such as antiviral porphyrins, with the ability to fight brain-resident viruses causing diseases such as HIV-associated neurocognitive disorders (HAND). In the last two decades, BBB shuttles, particularly peptide-based ones, have shown promise in carrying various payloads across the BBB. Thus, peptide–drug conjugates (PDCs) formed by covalent attachment of a BBB peptide shuttle and an antiviral drug may become key therapeutic tools in treating neurological disorders of viral origin. In this study, we have used various approaches (guanidinium, phosphonium, and carbodiimide-based couplings) for on-resin synthesis of new peptide–porphyrin conjugates (PPCs) with BBB-crossing and potential antiviral activity. After careful fine-tuning of the synthetic chemistry, DIC/oxyma has emerged as a preferred method, by which 14 different PPCs have been made and satisfactorily characterized. The PPCs are prepared by coupling a porphyrin carboxyl group to an amino group (either N-terminal or a Lys side chain) of the peptide shuttle and show effective in vitro BBB translocation ability, low cytotoxicity toward mouse brain endothelial cells, and low hemolytic activity. Three of the PPCs, MP-P5, P4-MP, and P4-L-MP, effectively inhibiting HIV infectivity in vitro, stand out as most promising. Their efficacy against other brain-targeting viruses (Dengue, Zika, and SARS-CoV-2) is currently under evaluation, with preliminary results confirming that PPCs are a promising strategy to treat viral brain infections., Work supported by the La Caixa Health Foundation (project HR17_00409, ID 100010434, agreement LCF/PR/HR17/ 52150011), by the European Union (H2020-FETOPEN-2018- 2019-2020-01 grant no 828774), and by the Spanish Ministry of Science and Innovation (AEI/FEDER grant CTQ2017- 84371-P). NMR experiments were performed in the “Manuel Rico” NMR laboratory, LMR, CSIC, a node of the Spanish Large-Scale National Facility ICTS R-LRB. Additional funding from Fundaca̧ o para a Cie ̃ ncia e Tecnologia (FCT-MCTES) is ̂ also acknowledged for DA Mendonca (PD/BD/136752/2018) ̧ and for C. Cruz-Oliveira and I. Cadima-Couto (PTDC/BIAVIR/29495/2017).