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In Chagas disease (ChD) caused by Trypanosoma cruzi, new biomarkers to predict chronic cardiac pathology are urgently needed. Previous studies in chagasic patients with mild symptomatology showed that antibodies against the immunodominant R3 epitope of sCha, a fragment of the human basic helix-loop-helix transcription factor like 5, correlated with cardiac pathology. To validate sCha as a biomarker and to understand the origin of anti-sCha antibodies, we conducted a multicenter study with several cohorts of chagasic patients with severe cardiac symptomatology. We found that levels of antibodies against sCha discriminated the high risk of sudden death, indicating they could be useful for ChD prognosis. We investigated the origin of the antibodies and performed an alanine scan of the R3 epitope. We identified a minimal epitope MRQLD, and a BLAST search retrieved several T. cruzi antigens. Five of the hits had known or putative functions, of which phosphonopyruvate decarboxylase showed the highest cross-reactivity with sCha, confirming the role of molecular mimicry in the development of anti-sCha antibodies. Altogether, we demonstrate that the development of antibodies against sCha, which originated by molecular mimicry with T. cruzi antigens, could discriminate electrocardiographic alterations associated with a high risk of sudden death.

Enzymatic cleavage of endogenous peptides is a commonly used principle to initiate, modulate and terminate action for instance among cytokines and peptide hormones. The incretin hormones, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1), and the related hormone glucagon-like peptide-2 (GLP-2) are all rapidly N-terminally truncated with severe loss of intrinsic activity. The most abundant circulating form of full length GIP(1-42) is GIP(3-42) (a dipeptidyl peptidase-4 (DPP-4) product). GIP(1-30)NH2 is another active form resulting from prohormone convertase 2 (PC2) cleavage of proGIP. Like GIP(1-42), GIP (1-30)NH2 is a substrate for DPP-4 generating GIP(3-30)NH2 which, compared to GIP(3-42), binds with higher affinity and very efficiently inhibits GIP receptor (GIPR) activity with no intrinsic activity. Here, we review the action of these four and multiple other N- and C-terminally truncated forms of GIP with an emphasis on molecular pharmacology, i.e. ligand binding, subsequent receptor activation and desensitization. Our overall conclusion is that the N-terminus is essential for receptor activation as GIP N-terminal truncation leads to decreased/lost intrinsic activity and antagonism (similar to GLP-1 and GLP-2), whereas the C-terminal extension of GIP(1-42), as compared to GLP-1, GLP-2 and glucagon (29-33 amino acids), has no apparent impact on the GIPR in vitro, but may play a role for other properties such as stability and tissue distribution. A deeper understanding of the molecular interaction of naturally occurring and designed GIP-based peptides, and their impact in vivo, may contribute to a future therapeutic targeting of the GIP system - either with agonists or with antagonists, or both.

Enzymatic cleavage of endogenous peptides is a commonly used principle to initiate, modulate and terminate action for instance among cytokines and peptide hormones. The incretin hormones, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1), and the related hormone glucagon-like peptide-2 (GLP-2) are all rapidly N-terminally truncated with severe loss of intrinsic activity. The most abundant circulating form of full length GIP(1-42) is GIP(3-42) (a dipeptidyl peptidase-4 (DPP-4) product). GIP(1-30)NH2 is another active form resulting from prohormone convertase 2 (PC2) cleavage of proGIP. Like GIP(1-42), GIP (1-30)NH2 is a substrate for DPP-4 generating GIP(3-30)NH2 which, compared to GIP(3-42), binds with higher affinity and very efficiently inhibits GIP receptor (GIPR) activity with no intrinsic activity. Here, we review the action of these four and multiple other N- and C-terminally truncated forms of GIP with an emphasis on molecular pharmacology, i.e. ligand binding, subsequent receptor activation and desensitization. Our overall conclusion is that the N-terminus is essential for receptor activation as GIP N-terminal truncation leads to decreased/lost intrinsic activity and antagonism (similar to GLP-1 and GLP-2), whereas the C-terminal extension of GIP(1-42), as compared to GLP-1, GLP-2 and glucagon (29-33 amino acids), has no apparent impact on the GIPR in vitro, but may play a role for other properties such as stability and tissue distribution. A deeper understanding of the molecular interaction of naturally occurring and designed GIP-based peptides, and their impact in vivo, may contribute to a future therapeutic targeting of the GIP system - either with agonists or with antagonists, or both.

Soil-transmitted helminths infect more than 2 billion people worldwide and only one drug (albendazole) is commonly available for use against intestinal parasite worms under conditions for mass drug administration. Recent studies have shown efficacy issues with this drug, stressing the importance of finding a new treatment option. Three-domain crystal (Cry) proteins produced from the soil bacterium Bacillus thuringiensis have been used for decades as a means to control insects that destroy crops and transmit human diseases, and studies have shown many of these proteins to be safe to humans. Our lab has shown that the crystal protein Cry5B is able to kill both the free-living nematode Caenorhabditis elegans, as well as parasitic roundworms (e.g., Ancylostoma ceylanicum, hookworm). Cry proteins intoxicate invertebrates by acting as pore-forming proteins. We believe that the nematode-Cry5B system has potential to unlock mysteries surrounding Cry proteins and to help improve Cry5B as a therapeutic agent. Here, I took a large scale systematic alanine-scanning approach and attempted to mutate all 698 amino acids in the toxin domain of Cry5B. I initially tested these mutants on C. elegans to assess for changes in toxicity levels, screening for variants with an increase in activity as compared to the wild type. Both the larger mutant set and the subset that had increased activity in my experiments are promising for future study, although the increased activity of the subset will need to be confirmed in assays with C. elegans and intestinal parasites. In the course of these studies, comparisons were made regarding the utility of CryB expressed in E coli, as purified protein, and as a spore/crystal mixture for activity assays and rescreening. Experiments were also carried out to address which system is best suited to test these variants in narrowing down those of interest and in predicting therapeutic potential in vivo. Overall, the Cry protein variants and approaches to their

Soil-transmitted helminths infect more than 2 billion people worldwide and only one drug (albendazole) is commonly available for use against intestinal parasite worms under conditions for mass drug administration. Recent studies have shown efficacy issues with this drug, stressing the importance of finding a new treatment option. Three-domain crystal (Cry) proteins produced from the soil bacterium Bacillus thuringiensis have been used for decades as a means to control insects that destroy crops and transmit human diseases, and studies have shown many of these proteins to be safe to humans. Our lab has shown that the crystal protein Cry5B is able to kill both the free-living nematode Caenorhabditis elegans, as well as parasitic roundworms (e.g., Ancylostoma ceylanicum, hookworm). Cry proteins intoxicate invertebrates by acting as pore-forming proteins. We believe that the nematode-Cry5B system has potential to unlock mysteries surrounding Cry proteins and to help improve Cry5B as a therapeutic agent. Here, I took a large scale systematic alanine-scanning approach and attempted to mutate all 698 amino acids in the toxin domain of Cry5B. I initially tested these mutants on C. elegans to assess for changes in toxicity levels, screening for variants with an increase in activity as compared to the wild type. Both the larger mutant set and the subset that had increased activity in my experiments are promising for future study, although the increased activity of the subset will need to be confirmed in assays with C. elegans and intestinal parasites. In the course of these studies, comparisons were made regarding the utility of CryB expressed in E coli, as purified protein, and as a spore/crystal mixture for activity assays and rescreening. Experiments were also carried out to address which system is best suited to test these variants in narrowing down those of interest and in predicting therapeutic potential in vivo. Overall, the Cry protein variants and approaches to their

Electrostatic interactions are ubiquitous in proteins and dictate stability and function. In this review, we discuss several methods for the analysis of electrostatics in protein-protein interactions. We discuss alanine-scanning mutagenesis, Poisson-Boltzmann electrostatics, free energy calculations, electrostatic similarity distances, and hierarchical clustering of electrostatic potentials. Our recently developed computational framework, known as Analysis of Electrostatic Similarities Of Proteins (AESOP), incorporates these tools to efficiently elucidate the role of electrostatic potentials in protein interactions. We present the application of AESOP to several proteins and protein complexes, for which charge is purported to facilitate protein association. Specifically, we illustrate how recent work has shaped the formulation of electrostatic calculations, the correlation of electrostatic free energies and electrostatic potential clustering results with experimental binding and activity data, the pH dependence of protein stability and association, the design of mutant proteins with enhanced immunological activity, and how AESOP can expose deficiencies in structural models and experimental data. This integrative approach can be utilized to develop mechanistic models and to guide experimental studies by predicting mutations with desired physicochemical properties and function. Alteration of the electrostatic properties of proteins offers a basis for the design of proteins with optimized binding and activity.

Electrostatic interactions are ubiquitous in proteins and dictate stability and function. In this review, we discuss several methods for the analysis of electrostatics in protein-protein interactions. We discuss alanine-scanning mutagenesis, Poisson-Boltzmann electrostatics, free energy calculations, electrostatic similarity distances, and hierarchical clustering of electrostatic potentials. Our recently developed computational framework, known as Analysis of Electrostatic Similarities Of Proteins (AESOP), incorporates these tools to efficiently elucidate the role of electrostatic potentials in protein interactions. We present the application of AESOP to several proteins and protein complexes, for which charge is purported to facilitate protein association. Specifically, we illustrate how recent work has shaped the formulation of electrostatic calculations, the correlation of electrostatic free energies and electrostatic potential clustering results with experimental binding and activity data, the pH dependence of protein stability and association, the design of mutant proteins with enhanced immunological activity, and how AESOP can expose deficiencies in structural models and experimental data. This integrative approach can be utilized to develop mechanistic models and to guide experimental studies by predicting mutations with desired physicochemical properties and function. Alteration of the electrostatic properties of proteins offers a basis for the design of proteins with optimized binding and activity.

Fallaxin is a 25-mer antibacterial peptide amide, which was recently isolated from the West Indian mountain chicken frog Leptodactylus fallax. Fallaxin has been shown to inhibit the growth of several Gram-negative bacteria including Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Here, we report a structure-activity study of fallaxin based on 65 analogs, including a complete alanine scan and a full set of N- and C-terminal truncated analogs. The fallaxin analogs were tested for hemolytic activity and antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-intermediate resistant S. aureus, (VISA), methicillin-susceptible S. aureus (MSSA), E. coli, K. pneumoniae, and P. aeruginosa. We identified several analogs, which showed improved antibacterial activity compared to fallaxin. Our best candidate was FA12, which displayed MIC values of 3.12, 25, 25, and 50 µM against E. coli, K. pneumoniae, MSSA, and VISA, respectively. Furthermore, we correlated the antibacterial activity with various structural parameters such as charge, hydrophobicity H" style="width: 4px; height: 15px; border-width: 0px"/>, mean hydrophobic moment µH" style="width: 4px; height: 15px; border-width: 0px"/>, and

Mycobacterium tuberculosis ribonucleotide reductase (RNR) is a potential target for new antitubercular drugs. Herein we describe the synthesis and evaluation of peptide inhibitors of RNR derived from the C-terminus of the small subunit of M. tuberculosis RNR. An N-terminal truncation, an alanine scan and a novel statistical molecular design (SMD) approach based on the heptapeptide Ac-Glu-Asp-Asp-Asp-Trp-Asp-Phe-OH were applied in this study. The alanine scan showed that TrP5 and Phe7 were important for inhibitory potency. A quantitative structure relationship (QSAR) model was developed based on the synthesized peptides which showed that a negative charge in positions 2, 3, and 6 is beneficial for inhibitory potency. Finally, in position 5 the model coefficients indicate that there is room for a larger side chain., as compared to Trp5.

Fallaxin is a 25-mer antibacterial peptide amide, which was recently isolated from the West Indian mountain chicken frog Leptodactylus fallax. Fallaxin has been shown to inhibit the growth of several Gram-negative bacteria including Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Here, we report a structure-activity study of fallaxin based on 65 analogs, including a complete alanine scan and a full set of N- and C-terminal truncated analogs. The fallaxin analogs were tested for hemolytic activity and antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-intermediate resistant S. aureus, (VISA), methicillin-susceptible S. aureus (MSSA), E. coli, K. pneumoniae, and P. aeruginosa. We identified several analogs, which showed improved antibacterial activity compared to fallaxin. Our best candidate was FA12, which displayed MIC values of 3.12, 25, 25, and 50 µM against E. coli, K. pneumoniae, MSSA, and VISA, respectively. Furthermore, we correlated the antibacterial activity with various structural parameters such as charge, hydrophobicity H" style="width: 4px; height: 15px; border-width: 0px"/>, mean hydrophobic moment µH" style="width: 4px; height: 15px; border-width: 0px"/>, and