Rapid and accurate structure-based therapeutic peptide design using GPU accelerated thermodynamic integration

Peptide-based therapeutics are an alternative to small molecule drugs as they offer superior specificity, lower toxicity, and easy synthesis. Here we present an approach that leverages the dramatic performance increase afforded by the recent arrival of GPU accelerated thermodynamic integration (TI). GPU TI facilitates very fast, highly accurate binding affinity optimization of peptides against therapeutic targets. We benchmarked TI predictions using published peptide binding optimization studies. Prediction of mutations involving charged side-chains was found to be less accurate than for non-charged, and use of a more complex 3-step TI protocol was found to boost accuracy in these cases. Using the 3-step protocol for non-charged side-chains either had no effect or was detrimental. We use the benchmarked pipeline to optimize a peptide binding to our recently discovered cancer target: EME1. TI calculations predict beneficial mutations using both canonical and non-canonical amino acids. We validate these predictions using fluorescence polarization and confirm that binding affinity is increased. We further demonstrate that this increase translates to a significant reduction in pancreatic cancer cell viability.     

Computational design of peptide ligands

Peptides possess several attractive features when compared to small molecule and protein therapeutics, such as high structural compatibility with target proteins, the ability to disrupt protein-protein interfaces, and small size. Efficient design of high-affinity peptide ligands via rational methods has been a major obstacle to the development of this potential drug class. However, structural insights into the architecture of protein-peptide interfaces have recently culminated in several computational approaches for the rational design of peptides that target proteins. These methods provide a valuable alternative to experimental high-resolution structures of target protein-peptide complexes, bringing closer the dream of in silico designed peptides for therapeutic applications.     

Synthesis of peptide analogues using the multipin peptide synthesis method

Modification of the multipin peptide synthesis method which allows the simultaneous synthesis of large numbers of different peptide analogues is described. Peptides were assembled on polyethylene pins derivatized with a 4-(beta-alanyloxymethyl)benzoate (beta-Ala-HMB) handle. For comparative purposes, peptides were also assembled on the diketopiperazine-forming handle N epsilon-(beta-alanyl)lysylprolyloxylactate. In model studies it was demonstrated that beta-Ala-HMB-linked peptides were cleaved from polyethylene pins with dilute sodium hydroxide or 4% methylamine/water to yield analogues with beta-Ala-free acid (beta-Ala-CO2H) and beta-Ala-methylamide (beta-Ala-CONHCH3), respectively. To assess the suitability of this approach for T-cell determinant analysis, analogues of a known T-cell determinant were synthesized with the various C-terminal endings. Peptides were characterized by amino acid analysis and fast atom bombardment-mass spectrometry. HPLC of the crude cleaved peptides indicated that 22 of the 24 peptides were greater than 95% pure. These crude peptide solutions were nontoxic in sensitive cell culture assays without further purification. All three cleavage procedures gave comparable activities in T-cell proliferation assays. These results demonstrate the potential of the multipin peptide synthesis method for the production of large numbers of different peptide analogues.     

Recognition characters in peptide–polyphenol complex formation

Dietary polyphenols have received attention for their anti-oxidative, anti-carcinogenic and anti-neurodegenerative effects. Polyphenols bind to proteins leading to the formation of soluble or insoluble protein–polyphenol complexes which could significantly influence their biological activities. NMR and molecular modeling studies were performed to investigate the influence of the bulk, flexibility and hydrophobicity of polyphenols on the association with bradykinin, the peptide model. Our results show that the strength of the interactions could be positively correlated with polyphenol hydrophobicity and a comparison between pentagalloylglucose and vescalagin indicated that flexibility might play a positive role in the interaction with peptides and proteins.     

Development and testing of protocols for computer-aided design of peptide drugs, using oxytocin

Considerable clinical interest in neuropeptides and peptide hormones has stimulated recent research and development of peptide-based drugs. This process differs from most classical drug discovery procedures because peptide molecules have considerable inherent flexibility. In the present paper, to identify lowest energy and metastable conformers for drug design, and to develop protocols for such studies, conformational search algorithms, incorporating empirical energy calculations, have been applied in the analysis of the peptide oxytocin. Energy minimization in torsion angle space was carried out from a variety of starting conformations, including published structures, in all-atom mode and all with distance constraints for disulphide bond formation. The energy-minimized conformations have been further optimized by a mapping method. Complementary simulations have been performed in united-atom mode and a model representing the effects of water using dummy sites has been developed and tested for this representation. Several of the preferred conformers together with de novo conformations have been used as starting points in molecular dynamics simulations; 28 low potential energy conformations were located at a temperature of 4 K. Conformations are analysed to identify hydrogen bonds, phi-psi angle distributions and the RMS values relative to the X-ray structure of deamino-oxytocin. The modelled structure of lowest energy in the molecular mechanics calculations was also that of least RMS deviation from the crystal structure; whilst structures of lower energy but larger deviation were identified by molecular dynamics techniques. A metastable structure has been identified which satisfies existing criteria for the "active form", and this model is tested by a theoretical residue-substitution technique, to provide clues on the agonist/antagonist relationship at the atomic level.     

Molecular Recognition Theory of the complementary (antisense) peptide interactions

Molecular Recognition Theory is based on the finding of Blalock et al. (Biochem. Biophys. Res. Commun. 121 (1984) 203–207; Nature Med. 1 (1995) 876–878; Biochem. J. 234 (1986) 679–683) that peptides specified by the complementary RNAs bind to each other with higher specificity and efficacy. This theory is investigated considering the interaction of the sense peptides coded by means of messenger RNA (read in 5′→3′ direction) and antisense peptides coded in 3′→5′ direction. We analysed the hydropathy of the complementary amino acid pairs and their frequencies in 10 peptide–receptor systems with verified ligand–receptor interaction. An optimization procedure aimed to reduce the number of possible antisense peptides derived from the sense peptide has been proposed. Molecular Recognition Theory was also validated by an “in vivo” experiment. It was shown that 3′→5′ peptide antisense of -MSH abolished its cytoprotective effects on the gastric mucosa in rats. Molecular Recognition Theory could be useful method to simplify experimental procedures, reduce the costs of the peptide synthesis, and improve peptide structure modelling.     

Template-assisted rational design of peptide inhibitors of furin using the lysine fragment of the mung bean trypsin inhibitor

Highly active, small-molecule furin inhibitors are attractive drug candidates to fend off bacterial exotoxins and viral infection. Based on the 22-residue, active Lys fragment of the mung bean trypsin inhibitor, a series of furin inhibitors were designed and synthesized, and their inhibitory activity towards furin and kexin was evaluated using enzyme kinetic analysis. The most potent inhibitor, containing 16 amino acid residues with a Ki value of 2.45x10(-9) m for furin and of 5.60x10(-7) m for kexin, was designed with three incremental approaches. First, two nonessential Cys residues in the Lys fragment were deleted via a Cys-to-Ser mutation to minimize peptide misfolding. Second, residues in the reactive site of the inhibitor were replaced by the consensus substrate recognition sequence of furin, namely, Arg at P1, Lys at P2, Arg at P4 and Arg at P6. In addition, the P7 residue Asp was substituted with Ala to avoid possible electrostatic interference with furin inhibition. Finally, the extra N-terminal and C-terminal residues beyond the doubly conjugated disulfide loops were further truncated. However, all resultant synthetic peptides were found to be temporary inhibitors of furin and kexin during a prolonged incubation, with the scissile peptide bond between P1 and P1' being cleaved to different extents by the enzymes. To enhance proteolytic resistance, the P1' residue Ser was mutated to D-Ser or N-methyl-Ser. The N-methyl-Ser mutant gave rise to a Ki value of 4.70x10(-8) m for furin, and retained over 80% inhibitory activity even after a 3 h incubation with the enzyme. By contrast, the d-Ser mutant was resistant to cleavage, although its inhibitory activity against furin drastically decreased. Our findings identify a useful template for the design of potent, specific and stable peptide inhibitors of furin, shedding light on the molecular determinants that dictate the inhibition of furin and kexin.     

抗原识别中的危险模式

Matzinger提出的危险模式摆脱了自我与非我模式(SNS)和感染的非我模式(INS)的束缚,认为免疫应答的启动是由损伤的细胞发出的危险信号所决定的,又组织细胞决定了免疫应答效应的类型。并运用危险信号模式对肿瘤、移植、母胎耐受、自身免疫性疾病、先天性淋巴细胞(NKIT细胞、YδT细胞)的作用等作了新的解释。     

Carbohydrates in peptide and protein design

Monosaccharides and amino acids are fundamental building blocks in the assembly of nature's polymers. They have different structural aspects and, to a significant extent, different functional groups. Oligomerization gives rise to oligosaccharides and peptides, respectively. While carbohydrates and peptides can be found conjoined in nature, e.g., in glycopeptides, the aim of this review is the radical redesign of peptide structures using carbohydrates, particularly monosaccharides and cyclic oligosaccharides, to produce novel peptides, peptidomimetics, and abiotic proteins. These hybrid molecules, chimeras, have properties arising largely from the combination of structural characteristics of carbohydrates with the functional group diversity of peptides. This field includes de novo designed synthetic glycopeptides, sugar (carbohydrate) amino acids, carbohydrate scaffolds for nonpeptidal peptidomimetics of cyclic peptides, cyclodextrin functionalized peptides, and carboproteins, i.e., carbohydrate-based proteinmimetics. These successful applications demonstrate the general utility of carbohydrates in peptide and protein architecture.     

合成肽疫苗的分子设计

合成肽疫苗能克服常规疫苗的缺点,很早就被认为是动物传染病预防用的终极疫苗。然而多年的研究结果表明,合成肽疫苗免疫动物后所起的免疫保护作用并没有象人们当初设想的那样理想,同时证明了构建的合成肽疫苗的抗原性及其免疫原性要受到其自身组成及宿主免疫系统等多种因素的影响。在诱导机体产生免疫的过程中,单一的中和抗原表位是远远不够的,增加中和抗原表位的数目和引入细胞抗原表位将起到必不可少的辅助协同作用。若想提高合成肽疫苗的免疫效果,在搞清合成肽疫苗的免疫机理并在如何利用有限的抗原表位诱导强有力的免疫保护作用等方面需要做进一步深入地研究。     

Recognition of double-stranded RNA by guanidine-modified peptide nucleic acids

Double-helical RNA has become an attractive target for molecular recognition because many noncoding RNAs play important roles in the control of gene expression. Recently, we discovered that short peptide nucleic acids (PNA) bind strongly and sequence selectively to a homopurine tract of double-helical RNA via formation of a triple helix. Herein, we tested if the molecular recognition of RNA could be enhanced by α-guanidine modification of PNA. Our study was motivated by the discovery of Ly and co-workers that the guanidine modification greatly enhances the cellular delivery of PNA. Isothermal titration calorimetry showed that the guanidine-modified PNA (GPNA) had reduced affinity and sequence selectivity for triple-helical recognition of RNA. The data suggested that in contrast to unmodified PNA, which formed a 1:1 PNA-RNA triple helix, GPNA preferred a 2:1 GPNA-RNA triplex invasion complex. Nevertheless, promising results were obtained for recognition of biologically relevant double-helical RNA. Consistent with enhanced strand invasion ability, GPNA derived from d-arginine recognized the transactivation response element of HIV-1 with high affinity and sequence selectivity, presumably via Watson-Crick duplex formation. On the other hand, strong and sequence selective triple helices were formed by unmodified and nucelobase-modified PNA and the purine-rich strand of the bacterial A-site. These results suggest that appropriate chemical modifications of PNA may enhance molecular recognition of complex noncoding RNAs.     

Investigating the effect of peptide agonists on the chondrogenic differentiation of human mesenchymal stem cells using design of experiments

Human mesenchymal stem cells (MSCs) are attractive for use in cartilage tissue engineering. Cells are often seeded in a structural scaffold containing growth factors. Peptide mimics of full‐length growth factors are a promising alternative because they are less expensive and easier to manufacture. We investigated four short peptides for their effect on the chondrogenesis of human MSCs. The peptides were originally designed to mimic bone morphogenetic protein‐2 (BMP‐2), transforming growth factor‐beta 1 (TGF‐β1), and insulin, all of which have been shown to affect MSC chondrogenesis. Previous studies demonstrated that the peptides elicited bioactivity in other cell types, but the peptides have not been investigated for their effect on chondrogenesis in human MSCs. In a preliminary investigation, peptides were added to a pellet culture of human MSCs and assayed for their effect on glycosaminoglycan (GAG) production. These experiments determined peptide concentrations used in a full‐factorial experiment to investigate any interactions. The experiment revealed the BMP peptide as a robust stimulant for GAG production. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1550–1557, 2013     

Angiotensin II inhibitory peptide found in the receptor sequence using peptide array

Peptide array consisting of hundreds of peptides spatially addressed and synthesized on a cellulose membrane support was used to screen ligand-inhibitory peptides. As a model, angiotensin II (Ang II), a significant peptide related to the treatment of cardiovascular diseases, was chosen as the target ligand. Peptide arrays covering the Ang II receptor type 1 sequence were prepared, and peptide domains with high affinity to the Ang II fluorescein conjugate were investigated. The peptide (VVIVIY) within the first transmembrane region exhibited the highest affinity to Ang II. The synthesized soluble VVIVIY peptide had an 84% inhibitory effect on Ang II-induced aorta contraction. These results indicate that our screening strategy utilizing peptide array is an effective approach for the peptide drug development.     

Exploitation of the selectivity-conferring code of nonribosomal peptide synthetases for the rational design of novel peptide antibiotics

Recently, the solved crystal structure of a phenylalanine-activating adenylation (A) domain enlightened the structural basis for the specific recognition of the cognate substrate amino acid in nonribosomal peptide synthetases (NRPSs). By adding sequence comparisons and homology modeling, we successfully used this information to decipher the selectivity-conferring code of NRPSs. Each codon combines the 10 amino residues of a NRPS A domain that are presumed to build up the substrate-binding pocket. In this study, the deciphered code was exploited for the first time to rationally alter the substrate specificity of whole NRPS modules in vitro and in vivo. First, the single-residue Lys239 of the L-Glu-activating initiation module C-A(Glu)-PCP of the surfactin synthetase A was mutated to Gln239 to achieve a perfect match to the postulated L-Gln-activating binding pocket. Biochemical characterization of the mutant protein C-A(Glu)-PCP(Lys239 --> Gln) revealed the postulated alteration in substrate specificity from L-Glu to L-Gln without decrease in catalytic efficiency. Second, according to the selectivity-conferring code, the binding pockets of L-Asp and L-Asn-activating A domains differs in three positions: Val299 versus Ile, His322 versus Glu, and Ile330 versus Val, respectively. Thus, the binding pocket of the recombinant A domain AspA, derived from the second module of the surfactin synthetases B, was stepwisely adapted for the recognition of L-Asn. Biochemical characterization of single, double, and triple mutants revealed that His322 represents a key position, whose mutation was sufficient to give rise to the intended selectivity-switch. Subsequently, the gene fragment encoding the single-mutant AspA(His322 --> Glu) was introduced back into the surfactin biosynthetic gene cluster. The resulting Bacillus subtilis strain was found to produce the expected so far unknown lipoheptapeptide [Asn(5)]surfactin. This indicates that site-directed mutagenesis, guided by the selectivity-conferring code of NRPS A domains, represents a powerful alternative for the genetic manipulation of NRPS biosynthetic templates and the rational design of novel peptide antibiotics.     

Structural basis for the design of antibiotics targeting peptide deformylase

While protein synthesis in bacteria begins with a formylated methionine, the formyl group of the nascent polypeptide is removed by peptide deformylase. Since eukaryotic protein synthesis does not involve formylation and deformylation at the N-terminus, there has been increasing interest in peptide deformylase as a potential target for antibacterial chemotherapy. Toward this end and to aid in the design of effective antibiotics targeting peptide deformylase, the structures of the protein-inhibitor complexes of both the cobalt and the zinc containing Escherichia coli peptide deformylase bound to the transition-state analogue, (S)-2-O-(H-phosphonoxy)-L-caproyl-L-leucyl-p-nitroanilide (PCLNA), have been determined. The proteins for both deformylase-inhibitor complexes show basically the same fold as for the native enzyme. The PCLNA inhibitor adopts an extended conformation and fits nicely into a hydrophobic cavity located near the metal site. On the basis of these structures, guidelines for the design of high-affinity deformylase inhibitors are suggested. As our results show that the protein residues which interact with the PCLNA inhibitor are conserved over a wide variety of species, we suggest that antibiotics targeting deformylase could have wide applicability.