PNA-encoded protease substrate microarrays

Our current understanding of the role and regulation of protease activity in normal and pathogenic processes is limited by our ability to measure and deconvolute their enzymatic activity. To address this limitation, an approach was developed that utilizes rhodamine-based fluorogenic substrates encoded with PNA tags. The PNA tags address each of the substrates to a predefined location on an oligonucleotide microarray through hybridization, thus allowing the deconvolution of multiple signals from a solution. A library of 192 protease substrates was prepared by split and mix combinatorial synthesis. The methodology and validation of this approach for profiling proteolytic activity from single proteases and from those in crude cell lysates as well as clinical blood samples is described.     

Selective hydrolysis of peptides promoted by metal ions: a positional scanning approach

Rapid, microplate-based fluorogenic screening of a positional scanning combinatorial library accurately predicts the sequence specificity of metal-assisted peptide hydrolysis.     

Solid-phase combinatorial library of norstatine-type isosters by the nitroaldol reaction

A combinatorial library of norstatine-type peptide isosters as putative inhibitors of aspartic proteases is presented. The library was synthesized using a split-and-mix strategy designed to afford a one-bead-two-compounds library with the isosteric elements positioned centrally in peptide chains. Application of ladder synthesis during library generation enabled structure identification by MALDI-TOF mass spectroscopy. The library was screened against aspartic protease renin, and two types of inhibitors were identified, that is, XXX-psi[CHRCHOH)-XXX and an aldehyde arising from unreacted starting material. Selected hits were resynthesized and assayed in solution, revealing inhibitors of nanomolar potency.     

Peptide microarrays for the determination of protease substrate specificity

A method is described for the preparation of substrate microarrays that allow for the rapid determination of protease substrate specificity. Peptidyl coumarin substrates, synthesized on solid support using standard techniques, are printed onto glass slides using DNA microarraying equipment. The linkage from the peptide to the slide is formed through a chemoselective reaction, resulting in an array of uniformly displayed fluorogenic substrates. The arrays can be treated with proteases to yield substrate specificity profiles. Standard instrumentation for visualization of microarrays can be used to obtain comparisons of the specificity constants for all of the prepared substrates. The utility of these arrays is demonstrated by the selective cleavage of preferred substrates with trypsin, thrombin, and granzyme B, and by assessing the extended substrate specificity of thrombin using a microarray of 361 different peptidyl coumarin substrates.     

Directed evolution of protease beacons that enable sensitive detection of endogenous MT1-MMP activity in tumor cell lines

Directed evolution was applied to identify peptide substrates with enhanced hydrolysis rates by MT1-MMP suitable for protease beacon development. Screening of a random pentapeptide library, using two-color CLiPS, yielded several substrates identical to motifs in distinct collagens that shared the consensus sequence P-x-G↓L. To identify substrates with enhanced cleavage rates, a second-generation decapeptide library incorporating the consensus was screened under stringent conditions, which resulted in a MxPLG↓(M)/(L)M(G)/(A)R consensus motif. These substrates are hydrolyzed by human-MT1-MMP up to six times faster than reported peptide substrates and are stable in plasma. Finally, incubation of soluble protease beacons incorporating the optimized substrates, but not previous substrates, enabled direct detection of endogenous MT1-MMP activity of human-fibrosarcoma (HT-1080) cells. Extended substrate libraries coupled with CLiPS should be useful to generate more effective activity probes for a variety of proteolytic enzymes.     

Combinatorial library of peptide isosters based on Diels-Alder reactions: identification of novel inhibitors against a recombinant cysteine protease from Leishmania mexicana

A combinatorial split-and-mix library of peptide isosters based on a Diels-Alder reaction was synthesized as a "one-bead-two-compounds" library and encoded by ladder synthesis for facile analysis by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. In the "one-bead-two-compounds" library approach, each bead contains a library member as a putative protease inhibitor along with a fluorescence-quenched substrate for the protease. When the library was screened with CPB2.8 DeltaCTE, a recombinant cysteine protease from L. mexicana, several beads containing compounds with inhibitory activity could be selected from the library and analyzed by MALDI-TOF MS for structure elucidation. Two types of inhibitors were revealed. One novel class of inhibitors had the bicyclic Diels-Alder product isosteric element incorporated internally in a peptide, while the other type was an N-terminal alpha,beta-unsaturated ketone Michael acceptor used as starting material for the Diels-Alder reaction. Selected hit sequences and constructed consensus sequences based on the observed frequencies of amino acids in different subsites were resynthesized and assayed in solution for inhibitor activity and were shown to have IC(50) values in the high nanomolar to low micromolar range.     

Development of protein, peptide, and small molecule catalysts using catalysis-based selection strategies

We have developed peptide catalysts and antibody catalysts that catalyze aldol, retro-aldol, and Michael reactions via an enamine mechanism using reaction-based selections with 1,3-diketone derivatives. Nucleophilic amino groups of the catalysts were covalently trapped during the selections. We have also developed fluorogenic substrates that are useful for real-time monitoring of the progress of bond-forming reactions, such as aldol reactions, by an increase in fluorescence. These fluorogenic substrates have been used to monitor peptide-catalyzed, antibody-catalyzed, enzyme-catalyzed, and small molecule-catalyzed reactions. Catalysis-based screening using fluorogenic substrates will accelerate rapid identification of superior catalysts and reaction conditions.     

Identification of highly reactive sequences for PLP-mediated bioconjugation using a combinatorial peptide library

Chemical reactions that facilitate the attachment of synthetic groups to proteins are useful tools for the field of chemical biology and enable the incorporation of proteins into new materials. We have previously reported a pyridoxal 5'-phosphate (PLP)-mediated reaction that site-specifically oxidizes the N-terminal amine of a protein to afford a ketone. This unique functional group can then be used to attach a reagent of choice through oxime formation. Since its initial report, we have found that the N-terminal sequence of the protein can significantly influence the overall success of this strategy. To obtain short sequences that lead to optimal conversion levels, an efficient method for the evaluation of all possible N-terminal amino acid combinations was needed. This was achieved by developing a generalizable combinatorial peptide library screening platform suitable for the identification of sequences that display high levels of reactivity toward a desired bioconjugation reaction. In the context of N-terminal transamination, a highly reactive alanine-lysine motif emerged, which was confirmed to promote the modification of peptide substrates with PLP. This sequence was also tested on two protein substrates, leading to substantial increases in reactivity relative to their wild-type termini. This readily encodable tripeptide thus appears to provide a significant improvement in the reliability with which the PLP-mediated bioconjugation reaction can be used. This study also provides an important first example of how synthetic peptide libraries can accelerate the discovery and optimization of protein bioconjugation strategies.     

Combinatorial Peptide Library for Probing the Selectivity of the s-1 Subsite of Proteases

A combinatorial tetrapeptide library, Suc-Ala-Phe-Arg-AA₁-OR, in which R = p-formamidobenzyl ester and AA₁ = 17 of the 20 natural occurring amino acids, has been synthesized chemically and separated by a reverse phase HPLC. The library was used to study the s-1 subsite specificity of various proteases. The preferred substrate at the s-1 subsite of chymotrypsin is in the order of Trp > Tyr > Phe > Met > Leu. This agreed with the reported data that the favored substrate at the s-1 subsite for chymotrypsin-catalyzed hydrolysis is an aromatic amino acid residue. The hydrophobic amino acid residues at this subsite can be hydrolysized after a longer incubating time. This procedure of selective hydrolysis of a peptide library was used to probe the selectivity of s-1 subsites of four proteases isolated from Bacillus stearothermophilus, subtilisin Carlsberg, subtilisin BPN' and an engineered protease subtilisin 8397. The protease from Bacillus stearothermophilus favored the substrate with residue Lys, and Arg at the s-1 subsite as a trypsin-like protease. The relative reactivities of amino acid residues in the protease-catalyzed hydrolysis of the library can be used as a fingerprint to identify the protease in a protease family.     

Solid phase combinatorial library of phosphinic peptides for discovery of matrix metalloproteinase inhibitors

A solid phase combinatorial library of 165,000 phosphinic peptide inhibitors was prepared and screened for activity against MMP-12. The inhibitors of the library had the structure XXX-Gpsi(PO2H-CH2)L-XXX, in which X is an arbitrary amino acid and Gpsi(PO2H-CH2)L is a Gly-Leu phosphinic dipeptide analogue. The library was constructed as a one-bead-two-compounds library so that every bead contained a common quenched fluorogenic substrate and a different putative inhibitor. In addition, the inhibitor part was prepared by ladder synthesis. After incubation with MMP-12, beads containing active inhibitors were selected, and the inhibitor sequences were recorded using MALDI-TOF MS. Statistical analysis of the sequences obtained from 86 beads gave rise to a consensus sequence which was resynthesized along with 20 related sequences. Three truncated sequences and 16 sequences originally present on beads were also resynthesized. The inhibitors were investigated in an enzyme kinetic assay with MMP-12 showing that the compounds derived from the consensus sequence were strong inhibitors with Ki values down to 6 nM, whereas the sequences originally present on beads varied in potency with Ki values from micromolar to nanomolar. Truncated sequences derived from the consensus sequence were poor inhibitors of MMP-12.     

High-throughput sequence determination of cyclic peptide library members by partial Edman degradation/mass spectrometry

Cyclic peptides provide attractive lead compounds for drug discovery and excellent molecular probes in biomedical research. Large combinatorial libraries of cyclic peptides can now be routinely synthesized by the split-and-pool method and screened against biological targets. However, post-screening sequence determination of hit peptides has been problematic. In this report, a high-throughput method for the sequence determination of cyclic peptide library members has been developed. TentaGel microbeads (90 mum) were spatially segregated into outer and inner layers; cyclic peptides were displayed on the bead surface, whereas the inner core of each bead contained the corresponding linear peptide as the encoding sequence. After screening of the cyclic peptide library against a macromolecular target, the identity of hit peptides was determined by sequencing the linear encoding peptides inside the bead using a partial Edman degradation/mass spectrometry method. On-bead screening of an octapeptide library (theoretical diversity of 160 000) identified cyclic peptides that bind to streptavidin. A 400-member library of tyrocidine A analogues was synthesized on TentaGel macrobeads and solution-phase screening of the library directly against bacterial cells identified a tyrocidine analogue of improved antibacterial activity. Our results demonstrate that the new method for cyclic peptide sequence determination is reliable, operationally simple, rapid, and inexpensive and should greatly expand the utility of cyclic peptides in biomedical research.     

Differential isotopic mass splitting as a mass spectrometric tool for identifying protease substrates

A method is described whereby stable isotopic signatures were partially incorporated into both termini of a peptide sequence giving rise to a characteristic cluster of four peaks in the mass spectral analysis. Cleavage of this peptide by a protease between the labeled positions generates two fragments both displaying their own individual signature peaks. The event of protease cleavage of the peptide was monitored by the changes in clusters within the spectrum. We believe that this technique could be used to aid the discovery of new cleavage substrates for proteases. Additionally, the analysis can be automated with dedicated software designed to select and interpret the data since all peaks of interest contain predefined signatures and can be easily distinguished from background noise.     

Identification of protease substrates by combinatorial profiling on TentaGel beads

Reacting a 65,536 member combinatorial library of octapeptides on TentaGel beads with various proteases followed by selective staining of the free amino termini at the reacted bead surface and sequence determination by amino acid analysis allowed the rapid identification of protease substrates.     

Fluorogenic peptide sequences--transformation of short peptides into fluorophores under ambient photooxidative conditions

Long-lived proteins are susceptible to nonenzymatic chemical reactions and the evolution of fluorescence; however, little is known about the sequence-dependence of fluorogenesis. We synthesized a library of over half a million octapeptides and exposed it to light and air in pH 7.4 buffer to identify fluorogenic peptides that evolve under mild oxidative conditions. The bead-based peptide library was composed of the general sequence H(2)N-Ala-(Xxx)(6)-Ala-resin, where Xxx was one of nine representative amino acids: Asp, Gly, His, Leu, Lys, Pro, Ser, Trp, and Tyr. Next, we selected five highly fluorescent beads from the library and subjected them to microsequencing, revealing the sequence of the unreacted peptide. All five of the fluorogenic sequences were ionic; lacked Tyr, His, and Leu; and most of the sequences contained only one Trp. We then synthesized the five soluble peptides corresponding to the fluorogenic peptide sequences and exposed them to photooxidative conditions. In general, the soluble peptides reacted slowly, generating nonfluorescent monooxygenated and dioxygenated products. However, one peptide (H(2)N-AlaLysProTrpGlyGlyAspAla-CONH(2)) evolved into a highly fluorescent photoproduct as well as a nonfluorescent monooxygenated photoproduct. The fluorescent photoproduct consisted of a 2-carboxy-quinolin-4-yl moiety fused to the N-terminus of GlyGlyAspAla. The formation of this photoproduct requires cleavage of the peptide backbone and a dramatic reorganization of tryptophan. This work demonstrates that sequencing unreacted peptide on beads can reveal sequences with unique nonenzymatic reactivity. The study also confirms that peptide fluorogenesis is dependent on sequence and not merely on the presence of tryptophan. The potential importance of fluorogenic peptide sequences is two-fold. First, fluorogenic sequences that arise through mutation could prove to be hot spots for human aging. Second, fluorogenic sequences, particularly those compatible with intracellular conditions, may serve as fluorescent tags for proteins or as fluorescent biomaterials.     

Expedient solid-phase synthesis of fluorogenic protease substrates using the 7-amino-4-carbamoylmethylcoumarin (ACC) fluorophore

A highly efficient solid-phase synthesis method for the preparation of fluorogenic protease substrates based upon the bifunctional leaving group 7-amino-4-carbamoylmethylcoumarin (ACC) is reported. Methods for the large-scale preparation of the novel fluorogenic leaving-group ACC are provided (Scheme 1). Detailed procedures are also provided for loading a diverse set of amino acids to support-bound ACC in good yields and with minimal racemization. Finally, procedures are included for the preparative synthesis of optimized ACC substrates for HIV-1 protease and plasmin.     

含7—氨基—4—甲基香豆素的蛋白酶荧光底物

本文设计并合成了一系列含７－氨基－４－甲基香豆素的肽类荧光底物，通过动力学测定研究了氨基酸结构对底物专一性的影响，发现Ｓｕｃ－Ａｌａ－Ａｌａ－Ｐｈｅ－ＡＭＣ和Ｓｕｃ－Ａｌａ－Ｐｒｏ－Ｐｈｅ－ＡＭＣ是较好的胰凝乳蛋白酶荧光底物。     

Fluorescence-quenched solid phase combinatorial libraries in the characterization of cysteine protease substrate specificity

To map the substrate specificity of cysteine proteases, two combinatorial peptide libraries were synthesized and screened using the archetypal protease, papain. The use of PEGA resin as the solid support for library synthesis facilitated the application of an on-resin fluorescence-quenched assay. Results from the screening of library 2 indicated a preference for Pro or Val in the S3 subsite and hydrophobic residues in S2; the most prevalent residue not being Phe but Val. The S1 subsite exhibited a dual specificity for both small, nonpolar residues, Ala or Gly, as well as larger, Gln, and charged residues, Arg. Small residues predominated in the S1'-S4' subsites. Active peptides from the libraries and variations thereof were resynthesized and their kinetics of hydrolysis by papain assessed in solution phase assays. Generally, there was a good correlation between the extent of substrate cleavage on solid phase and the kcat/KM's obtained in solution phase assays. Several good substrates for papain were obtained, the best substrates being Y(NO2)PMPPLCTSMK(Abz) (kcat/KM = 2109 (mM s)-1), Y(NO2)PYAVQSPQK(Abz) (kcat/KM = 1524 (mM s)-1), and Y(NO2)PVLRQQRSK(Abz) (kcat/KM = 1450 (mM s)-1). These results were interpreted in structural terms by the use of molecular dynamics (MD). These MD calculations indicated two different modes for the binding of substrates in the narrow enzyme cleft.     

Activity profile of dust mite allergen extract using substrate libraries and functional proteomic microarrays

Enzymatic activity in the fecal droppings from the house dust mite has been postulated to contribute to the elicited allergic response. Screening dust mite extracts through 137,180 tetrapeptide fluorogenic substrates allowed for the characterization of proteolytic substrate specificity from the potential cysteine and serine proteases in the extract. The extract was further screened against a 4000 member peptide nucleic acid (PNA) encoded inhibitor library designed to target cysteine proteases using microarray detection. Affinity chromatography coupled with mass spectrometry identified Der p 1 as one of the proteases targeted by the PNA inhibitors in the dust mite lysate. A phenotypic readout of Der p 1 function in allergy progression was demonstrated by the inhibition of CD25 cleavage from T cells by dust mite extract that had been treated with the Der p 1 inhibitor identified from the PNA-encoded inhibitor library.     

A Mechanism‐Based Approach to Screening Metagenomic Libraries for Discovery of Unconventional Glycosidases

Functional metagenomics has opened new opportunities for enzyme discovery. To exploit the full potential of this new tool, the design of selective screens is essential, especially when searching for rare enzymes. To identify novel glycosidases that employ cleavage strategies other than the conventional Koshland mechanisms, a suitable screen was needed. Focusing on the unsaturated glucuronidases (UGLs), it was found that use of simple aryl glycoside substrates did not allow sufficient discrimination against β‐glucuronidases, which are widespread in bacteria. While conventional glycosidases cannot generally hydrolyze thioglycosides efficiently, UGLs follow a distinct mechanism that allows them to do so. Thus, fluorogenic thioglycoside substrates featuring thiol‐based self‐immolative linkers were synthesized and assessed as selective substrates. The generality of the approach was validated with another family of unconventional glycosidases, the GH4 enzymes. Finally, the utility of these substrates was tested by screening a small metagenomic library.