Extracellular proteases as targets for treatment of cancer metastases

Metastasis, the dissemination of tumor cells to distant organs, is often associated with fatal outcome in cancer patients. Formation of metastasis requires degradation of extracellular matrices and several families of proteases have been implicated in this process, including matrix metalloproteinases (MMPs), serine and cysteine proteases. Inhibition of these enzymes in animal models of metastasis has shown impressive therapeutic effects. This report discusses the various approaches used for enzyme inhibition and describes new developments in drug design for inhibition of proteases in metastatic disease.     

"In-gel patch electrophoresis:" a new method for environmental DNA purification

Most of the microorganism species are largely untapped and could represent an interesting reservoir of genes useful for biotechnological applications. Unfortunately, a major difficulty associated with the methods used to isolate environmental DNA is related to the contamination of the extracted material with humic substances. These polyphenolic compounds inhibit the DNA processing reactions and severely impede cloning procedures. In this work, we describe a rapid, simple, and efficient method for the purification of genomic DNA from environmental samples: we added a chromatography step directly embedded into an agarose gel electrophoresis. This strategy enabled the DNA extraction from various environmental samples and it appeared that the purity grade was compatible with digestion by restriction enzymes and polymerase chain reaction (PCR) amplifications.     

Polar Cycloadditions

Cycloadditions with ionic components are known as “polar cycloadditions” to distinguish them from cycloadditions with dipolar and uncharged components. The structural requirements, the manner in which “polar” intermediates capable of addition are produced and their reactivity with activated and nonactivated multiple bonds, the many peculiarities of the course of the cycloaddition, and the favorable synthesis of heterocyclic systems by this reaction principle makes such a terminological distinction useful and necessary if the existing data are to be brought into order. The present review, which is the first on a field of chemistry that has not yet been very extensively investigated, deals initially with cationic and then with anionic polar cycloadditions.     

"Click" synthesis of small molecule-peptide conjugates for organelle-specific delivery and inhibition of lysosomal cysteine proteases

A click chemistry approach for the synthesis of small molecule inhibitor-peptide conjugates to achieve organelle-specific delivery has been developed. Biological testing showed that the inhibitor-Tat conjugate was successfully delivered to the lysosomes, leading to potent inhibition of lysosomal cysteine proteases in cultured cells.     

Substrate phage as a tool to identify novel substrate sequences of proteases

Combinatorial phage peptide libraries have been used to identify the ligands for specific target molecules. These libraries are also useful for identification of the specific substrates of various proteases. A substrate phage library has a random peptide sequence at the N-terminus of the phage coat protein and an additional tag sequence that enables attachment of the phage to an immobile phase. When these libraries are incubated with a specific enzyme, such as a protease, the uncleaved phage is excluded from the solution with tag-binding macromolecules. This provides a novel approach to define substrate specificity. The aim of this review is to summarize recent progress on the application of the substrate phage technique to identify specific substrates of proteolytic enzymes. As an example, some of our own experimental data on the selection and characterization of substrate sequences for thrombin, a serine protease, and membrane type-1 matrix metalloproteinase (MT1-MMP) will be presented. Using this approach, the canonical consensus substrate sequence for thrombin was deduced from the selected clones. As expected from the collagenolytic activity of MT1-MMP, a collagen-like sequence was identified in the case of MT1-MMP. A more selective substrate sequence for MT1-MMP was identified during a substrate phage screen. The delineation of the substrate specificity of proteases will help to elucidate the enzymatic properties and the physiological roles of these enzymes. Comprehensive screening of very large numbers of potential substrate sequences is possible with substrate phage libraries. Thus, this approach allows novel substrate sequences and previously unknown target molecules to be defined.     

Cycloadditions with Polar Intermediates

A discussion of the criteria that have been used to distinguish experimentally between one-step and two-step cycloadditions shows that in most cases useful information can be obtained only by isolation or interception of intermediates; the isolation of intermediates is also of preparative importance.     

Plant waste residues as inducers of extracellular proteases for a deuteromycete fungus <Emphasis Type="Italic">Trichoderma atroviride</Emphasis>

The ability of Trichoderma atroviride F-534 to utilize plant waste byproducts derived from processing of vegetables and fruits, as the major source of organic carbon and nitrogen for growth and protease production, was tested. The submerged cultivation of T. atroviride F-534 in the mineral base of the Czapek–Dox medium supplemented with plant waste byproducts resulted into copious biomass formation and was accompanied by secretion of several proteolytic enzymes. Zymography analysis of fungal culture filtrates showed that the high-molecular weight (HMW) protease(s) (from 100 kDa to 230 kDa) represent the major portion of secreted enzymes. Serine-type proteases and metalloproteases were predominant, although all known types of proteolytic enzymes were detected dependent on the type of inducer (substrate). The most conspicuous feature of secreted proteases was that the zymography patterns were unique for each plant material tested. These results confirm our previous finding obtained with purified proteins. Results also suggest that HMW protease(s) may participate in the heterotrophic/saprophytic/mode of life of this fungus. Their identity remains, however, obscure.     

Polar Substituent Effects in the Solvolysis of Acyclic Tertiary Chlorides Polar Effects VI

The influence of twenty substituents R on the solvolysis rates and products of tertiary chlorides R - CH₂CH₂C(CH₃)₂Cl (7) has been studied. H, CH₃, (CH₃)₂NCH₂, ClCH₂, Cl, CN and NO₂ exert an inductive effect only, as the good correlation of the rates with the corresponding inductive substituent constants σ shows. Bulkier alkyl groups, i.e. isopropyl and t-butyl, lower the rate due to a Baker-Nathan effect, while the n-electron donors CH₃S, CH₃O, HO and (CH₃)₂N and the σ-electron donors (CH₃)₃Sn and HOCH₂ cause rate increases based on σ constants. These accelerations are attributed to C,C-hyperconjugative and inductomeric effects which arise in the transition state for ionization. A comparison of the reactivity of the acyclic chlorides 7 and the corresponding 1-R-substituted 3-bromoadamantanes 1a shows that polar substituent effects are more strongly transmitted in the rigid bicyclic compounds 1a than in the flexible acyclic compounds 7.     

Solid-phase synthesis of peptide vinyl sulfones as potential inhibitors and activity-based probes of cysteine proteases

Peptide vinyl sulfones were prepared from 2-chlorotrityl resin-bound phenolic amino vinyl sulfones in high yield and purity. This method enables the convenient synthesis of peptide vinyl sulfones having different amino acids at the P(1) position. It also allows efficient synthesis of vinyl sulfone-containing, activity-based probes of cysteine proteases used in a proteomic experiment.     

Immobilized proteases for wound cleaning

Data on the immobilization of proteolytic enzymes for creation of medicines for the first phase of wound healing are summarized. The most common methods of immobilization, media, and pharmaceutical forms are characterized.     

Chemically modified "polar patch" mutants of subtilisin in peptide synthesis with remarkably broad substrate acceptance: designing combinatorial biocatalysts

A significant enhancement of the applicability of the serine protease subtilisin Bacillus lentus (SBL) in peptide synthesis was achieved by using the strategy of combined site-directed mutagenesis and chemical modification to create chemically modified mutant (CMM) enzymes. The introduction of polar and/or homochiral auxiliary substituents, such as X=oxazolidinones, alkylammonium groups, and carbohydrates at position 166 at the base of the primary specificity S(1) pocket created SBL CMMs S166C-S-X with strikingly broad structural substrate specificities. These CMMs are capable of catalyzing the coupling reactions of not only L-amino acid esters but also D-amino acid esters as acyl donors with glycinamide to give the corresponding dipeptides in good yields. These powerful enzymes are also applicable to the coupling of L-amino acid acyl donors with alpha-branched acyl acceptor, L-alaninamide. Typical increases in isolated yields of dipeptides of 60-80 % over SBL-WT (e.g. 0 % yield of Z-D-Glu-GlyNH(2) using SBL-WT-->74 % using S166C-S-(CH(2))(2) NMe(3) (+)) demonstrate the remarkable synthetic utility of this "polar patch" strategy. Such wide-ranging systems displaying broadened and therefore similarly high, balanced yields of products (e.g. 91 % Z-L-Ala-GlyNH(2) and 86 % yield of Z-D-Ala-GlyNH(2) using S166C-S-(3R,4S)-indenooxazolidinone) may now allow the use of biocatalysts in parallel library synthesis.     

Polar Effects in Organic Reactions

Attempts to gauge the effect of polar substituents on organic reaction rates by means of “inductive substituent constants” are based on the assumption that these effects are independent of the type of reaction observed. The measured rate constants of nucleophilic substitution reactions show, however, that this assumption is only partly justified even for saturated molecules. It is invalid if the substituent and the reaction center are an electron donor and an electron acceptor, respectively, which are hyperconjugated by way of σ-bonds. In extreme cases the resulting polarization can lead to heterolytic fragmentation.