Non-natural Amino Acid Residues Research Articles

Design of Optical-Imaging Probes by Screening of Diverse Substrate Libraries Directly in Disease-Tissue Extracts.

Fluorescently quenched probes that are specifically activated in the cancer microenvironment have great potential application for diagnosis, early detection, and surgical guidance. These probes are often designed to target specific enzymes associated with diseases by direct optimization using single purified enzymes. However, this can result in painstaking chemistry efforts to produce a probe with suboptimal performance when applied in vivo. We describe here an alternate, unbiased activity-profiling approach in which whole tissue extracts are used to directly identify optimal peptide sequences for probe design. Screening of tumor extracts with a hybrid combinatorial substrate library (HyCoSuL) identified a combination of natural and non-natural amino-acid residues that was used to generate highly efficient tumor-specific probes. This new strategy simplifies and enhances the process of probe optimization without any a priori knowledge of enzyme targets and has the potential to be applied to diverse disease states using clinical or animal-model tissue samples.

Peptibodies: An elegant solution for a long-standing problem.

Chimeric proteins composed of a biologically active peptide and a fragment crystallizable (Fc) domain of immunoglobulin G (IgG) are known as peptibodies. They present an extended half-life due to neonatal Fc receptor (FcRn) salvage pathway, a decreased renal clearance rate owing to its increased size (≈70 kDa) and, depending on the peptide used in the design of the peptibody, an active-targeting moiety. Also, the peptides therapeutic activity is boosted by the number of peptides in the fusion protein (at least two peptides) and to some peptides' alterations. Peptibodies are mainly obtained through recombinant DNA technology. However, to improve peptide properties, "unnatural" changes have been introduced to the original peptides' sequence, for instance, the incorporation of D- or non-natural amino acid residues or even cyclization thus, limiting the application of genetic engineering in the production of peptibodies, since these peptides must be obtained via chemical synthesis. This constrains prompted the development of new methods for conjugation of peptides to Fc domains. Another challenge, subject of intense research, relates to the large-scale production of such peptibodies using these new techniques, which can be minimized by their proved value. To date, two peptibodies, romiplostim and dulaglutide, have been approved and stay as the standard of care in their areas of action. Furthermore, a considerable number of peptibodies are currently in preclinical and clinical development.

Introduction of non-natural amino acid residues into the substrate-specific P 1 position of trypsin inhibitor SFTI-1 yields potent chymotrypsin and cathepsin G inhibitors

A series of trypsin inhibitor SFTI-1compounds modified in substrate-specific P 1 position was synthesized by the solid-phase method. Lys5 present in the wild inhibitor was replaced by Phe derivatives substituted in para position of the phenyl ring, l-pyridylalanine and N-4 -nitrobenzylgycine. Their inhibitory activities with bovine α-chymotrypsin and cathepsin G were estimated by determination of association equilibrium constants ( K a). All analogues inhibited bovine α-chymotrypsin. The highest inihbitory activity displayed peptides with the fluorine, nitro and methyl substituents. They were 13–15-fold more active than [Phe 5]SFTI-1 used as a reference. They are the most potent chymotrypsin inhibitors of this size. Substitution of Lys5 by Phe did not change the cathepsin G inhibitory activity. Introduction of Phe( p-F), Phe( p-NH 2) and Phe( p-CH 3) in this position retained the affinity towards this proteinase, whereas Phe( p-guanidine) gave an inhibitor more than twice as active, which appeared to be stable in human serum. On the other hand, a peptomeric analogue with N-4-nitrobenzylglycine failed to inhibit cathepsin G. Despite the fact the introduced amino acids were non-coded, the peptide bonds formed by them were hydrolyzed by chymotrypsin. We postulate that additional interaction of para-substitutents with the enzyme are responsible for the enhanced inhibitory activity of the analogues.

Truncation and non-natural amino acid substitution studies on HTLV-I protease hexapeptidic inhibitors

The culprit behind adult T-cell leukemia, myelopathy/tropical paraparesis, and a plethora of inflammatory diseases is the human T-cell leukemia virus type 1 (HTLV-I). We recently unveiled a potent hexapeptidic HTLV-I protease inhibitor, KNI-10166, composed mostly of natural amino acid residues. Herein, we report the derivation of potent tetrapeptidic inhibitor KNI-10516, possessing only non-natural amino acid residues.

The first semi-synthetic serine protease made by native chemical ligation

Selective incorporation of non-natural amino acid residues into proteins is a powerful approach to delineate structure–function relationships. Although many methodologies are available for chemistry-based protein engineering, more facile methods are needed to make this approach suitable for routine laboratory practice. Here, we describe a new strategy and provide a proof of concept for engineering semi-synthetic proteins. We chose a serine protease Streptomyces griseus trypsin (SGT) for this study to show that it is possible to efficiently couple a synthetic peptide containing a catalytically critical residue to a recombinant fragment containing the other active site residues. The 223-residue hybrid SGT molecule was prepared by fusing a chemically synthesized N-terminal peptide to a large C-terminal fragment of recombinant origin using native chemical ligation. This C-terminal polypeptide was produced from full-length SGT by cyanogen bromide cleavage at a genetically engineered Met57 position. This semi-synthetic hybrid trypsin is fully active, showing kinetics identical to the wild-type enzyme. Thus, we believe that it is an ideal model enzyme for studying the catalytic mechanisms of serine proteases by providing a straightforward approach to incorporate non-natural amino acids in the N-terminal region of the protein. In particular, this strategy will allow for replacement of the catalytic His57 residue and the buried N-terminus, which is thought to help align the active site, with synthetic analogs. Our approach relies on readily available recombinant proteins and small synthetic peptides, thus having general applications in chemical engineering of large proteins where the N-terminal region is the focal interest.

Peptidomimetic design of CDK inhibitors targeting the recruitment site of the cyclin subunit.

The recognition of cyclin-dependent kinase (CDK)/cyclin complexes by various cell-cycle regulatory proteins, including certain tumour suppressors and transcription factors, occurs at least in part through a protein-protein interaction with a binding groove on the cyclin subunit. Since CDK function is generally deregulated in tumour cells, blocking of this recruitment site prevents recognition and subsequent phosphorylation of CDK substrates and offers a therapeutic approach towards restoration of checkpoint control in transformed cells. Here we discuss the finding that peptides derived from such cyclin-interacting proteins, and rendered permeable through conjugation to cellular delivery vectors, can apparently induce tumour cells to undergo apoptosis selectively. We review the current status of 3D-structural information available on cyclin-peptide interactions and we summarise our extensive peptide structure-activity relationship studies in light of this information. We also show how a combination of molecular modelling and introduction into synthetic peptides of peptidomimetic elements, such as non-natural amino acid residues and conformational constraints, is being used hopefully to arrive at drug candidates capable of modulating CDK function in a selective mechanism-based approach rather than through ATP antagonism.

Semisynthesis of ribonuclease A using intein-mediated protein ligation.

The introduction of non-natural amino acid residues or modules into proteins provides a new means to explore the basis for conformational stability, folding/unfolding behavior, or biological function. We exploited intein-mediated protein ligation to produce a semisynthetic ribonuclease A. Of the 124 residues of RNase A, residues 1–94 were linked to an intein. After expression of the fusion protein and thiol-induced cleavage, the RNase A(1–94) fragment possessed a C-terminal thioester. A peptide identical to the C-terminal residues 95–124 of RNase A (with residue 95 being cysteine) was successfully ligated to that thioester thereby reconstituting full-length wild-type RNase A. In mass spectrometry, this semisynthetic RNase A proved to be undistinguishable from the control protein, namely recombinant wild-type RNase A. Recombinant wild-type RNase A was obtained by expression of RNase A(1–124)–intein fusion protein followed by thiol-induced cleavage and hydrolysis of the thioester. Both proteins showed thermal stabilities (Tm ) and catalytic activities comparable to the wild-type enzyme, indicating that both proteins folded properly. These results might serve as basis for the semisynthesis of RNase A variants containing non-natural modules in the aforementioned peptide.

Biosynthesis of a highly stable coiled-coil protein containing hexafluoroleucine in an engineered bacterial host.

Incorporation of nonnatural amino acid residues allows engineering of proteins with novel chemical functionality and unusual physical properties. We have shown recently that coiled-coil proteins prepared in vivo can be stabilized significantly by replacement of leucine by trifluoroleucine (1). In the same series of experiments, however, we were unsuccessful in our attempts to incorporate the more highly fluorinated analogue hexafluoroleucine (2). We report here that modification of the leucyl-tRNA synthetase (LeuRS) activity of the host allows efficient incorporation of 2 into recombinant proteins prepared in Escherichia coli. Furthermore, the coiled-coil protein used to demonstrate incorporation of 2 exhibits enhanced stability in comparison to the same protein enriched in 1, possibly due to the increased hydrophobic character of the additional trifluoromethyl group in the protein core.

Semisynthesis of Protein variants Using Intein-Mediated Protein Ligation.

INTRODUCTION. The introduction of non-natural amino acid residues or modules into proteins enables the study of the significance of single amino acid residues or entire structural regions in the stability of the protein, its folding/unfolding behavior, or its biological function. Bovine pancreatic ribonuclease A (RNase A) is a small protein with a well-characterized structure and thoroughly studied stability and unfolding/folding behavior (1). More recently, RNase A also became a target of studies as a chemotherapeutic (2). We exploited the method of intein-mediated protein ligation (3) to introduce non-natural amino acid residues into the C-terminal part of RNase A and investigated the biochemical properties of the variants. METHOD. Peptides containing either the wild-type RNase A sequence Cys95-Val124 or the same sequence with replacements for certain amino acid residues were synthesized by solidphase methods. Full-length RNase A or RNase A 1-94 and variants of these were produced as a fusion protein. The respective DNA sequence was inserted into the plasmids pTYB1 and pTXB1 to produce a fusion protein, which consists of RNase A (1-124 or 1-94) with an additional Met –1, the intein, and a chitin-binding domain (4). RESULTS. With pTYB1, purification of the fusion protein and thiol-facilitated formation of a thioester at the C-terminal Asn94 of the RNase A 1-94 fragment resulted in high amounts of cleavage product. However, only a little fraction contained the required thioester due to the slow intein cleavage and the fast thioester hydrolysis. Ligation of the intein cleavage product with the corresponding peptide yielded about 70 µg of ligation product per liter of bacterial culture. An N94A RNase A 1-94 variant proved to result in considerable higher yields of thioester, enabling efficient ligation of the protein component with the peptide. Here, ligation yielded about 200 µg of product per liter of bacterial culture. With pTXB1, the yields of fusion protein were substantially higher which allowed a formation of ligation product in reasonable amounts.

Pentavalent Organo-Vanadates as Transition State Analogues for Phosphoryl Transfer Reactions.

Pentavalent organo-vanadates have been put forth as transition state analogues for a variety of phosphoryl transfer reactions. In particular, uridine 2',3'-cyclic vanadate (U>v) has been proposed to resemble the transition state during catalysis by ribonuclease A (RNase A). Here, this hypothesis is tested. Lys41 of RNase A is known to donate a hydrogen bond to a nonbridging phosphoryl oxygen in the transition state during catalysis. Site-directed mutagenesis and semisynthesis were used to create enzymes with natural and nonnatural amino acid residues at position 41. These variants differ by 10(5)-fold in their k(cat)/K(m) values for catalysis, but <40-fold in their K(i) values for inhibition of catalysis by U>v. Plots of logK(i) vs log(K(m)/k(cat)) for three distinct substrates [poly(cytidylic acid), uridine 3'-(p-nitrophenyl phosphate), and cytidine 2',3'-cyclic phosphate] have slopes that range from 0.25 and 0.36. These plots would have a slope of unity if U>v were a perfect transition state analogue. Values of K(i) for U>v correlate weakly with the equilibrium dissociation constant for the enzymic complexes with substrate or product, indicating that U>v bears some resemblance to the substrate and product as well as the transition state. Thus, U>v is a transition state analogue for RNase A, but only a marginal one. This finding indicates that a pentavalent organo-vanadate cannot necessarily be the basis for a rigorous analysis of the transition state for a phosphoryl transfer reaction.