Adam Lesner

Introduction of non-natural amino acid residues into the substrate-specific P1 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 alpha-chymotrypsin and cathepsin G were estimated by determination of association equilibrium constants (K(a)). All analogues inhibited bovine alpha-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.

A Soluble Factor Secreted by an HIV-1-Resistant Cell Line Blocks Transcription through Inactivating the DNA-Binding Capacity of the NF-κB p65/p50 Dimer

The identity and activity of several anti-HIV soluble factor(s) secreted by CD8 and CD4 T lymphocytes have been determined; however, some of them still await definition. We have established an HIV-1-resistant, transformed CD4 T cell line that secretes HIV-1 resistance protein(s). Our studies indicate that this protein(s), called HIV-1 resistance factor (HRF), inhibits transcription of the virus by interfering with the activity of NF-κB. In the present report we identified the site at which HRF exerts this inhibition by evaluating a set of discrete events in NF-κB action. We tested the κB oligonucleotide binding activity in nuclei of resistant cells, nuclear translocation and binding to the HIV-1 long terminal repeat of p65 and p50 proteins from susceptible cells after exposure to HRF, and the binding of recombinant p50 to the κB oligonucleotide in vitro as affected by prior or simultaneous exposure to HRF. The results of this experimental schema indicate that HRF interacts with p50 after it enters the nucleus, but before its binding to DNA and that this interaction impedes the formation of an NF-κB-DNA complex required for the promotion of transcription. These findings suggest that HRF mediates a novel innate immune response to virus infection.

New potent cathepsin G phosphonate inhibitors

Cathepsin G is an enzyme with dual chymotrypsin and trypsin-like specificity. As a leukocyte proteinase it is involved in the early stages of the immune response. In this work the synthesis and inhibitory activity of diaryl phosphonic-type irreversible cathepsin G inhibitors are described. Modification of the lead structure Z-Phg(P)(OPh)2 (k(obs)/I=91 M(-1)s(-1)) in phenyl ester moieties followed by incorporation of the basic functional group into the aromatic side chain yielded highly potent cathepsin G inhibitor Z-(4-guanidine)Phg(P)(OC6H4-4-S-Me)2 with the apparent second-order inhibition value at 15,600 M(-1)s(-1). Further elongation of the obtained compound by tripeptide resulted in the inhibitor Ac-Phe-Val-Thr-(4-guanidine)Phg(P)(OC6H4-4-S-Me)2 with the highest k(obs)/I value ever reported in literature (256,000 M(-1)s(-1)).

Inhibition of HIV-1 or bacterial activation of macrophages by products of HIV-1-resistant human cells

We have recently described the molecular basis of HIV‐1 resistance factor (HRF)‐mediated anti‐viral activity in primary and transformed CD4 T cells. HRF(+) cell culture supernatants or partially purified HRF were found to incapacitate the formation of the NF‐κB/DNA complex, which is indispensable for long terminal promoter‐driven transcription of virus genes. In this study, we tested whether HRF might have much broader activity against other organisms whose pathogenesis is linked to NF‐κB activation. Specifically, we tested the effects of HRF on the NF‐κB‐mediated responses of primary macrophages to HIV‐1 or several bacterial antigens. We found that exposure to HRF inhibited HIV‐1 expression in macrophages and also induced the production of HRF‐like activity by macrophages, which prevented replication of virus in HIV‐1‐infected peripheral blood lymphocytes cultured in the adjacent compartment. We investigated the mechanism of this inhibition and found that HRF impeded NF‐κB/DNA binding in macrophages induced by either HIV‐1 or lipopolysaccharide from several bacteria species, resulting in impaired tumor necrosis factor‐alpha responses to these organisms.

Selection of New Chromogenic Substrates of Serine Proteinases Using Combinatorial Chemistry Methods

Chemical synthesis, physicochemical characterization and kinetic investigations of a tetrapeptide library of chromogenic substrates containing the amide of 5-amino-2-nitrobenzoic acid (Anb(5,2)-NH(2)) at their C-termini are reported. Anb(5,2)-NH(2) served as a chromophore released upon enzymatic action. The library consisting of 9567 peptides was synthesized using the portioning-mixing method and was screened against bovine a-chymotrypsin and human leukocyte elastase in solution applying an iterative approach. The selected chromogenic substrates were resynthesized and further modified at their N- and C-termini. Finally, two sequences, Z-Phe-Ala-Thr-Tyr-Anb(5,2)-NH(2) and Z-Phe-Phe-Pro-Val-Anb(5,2)-NH(2), were obtained as highly specific substrates for bovine alpha-chymotrypsin and human leukocyte elastase, respectively. The method of synthesis and selection of chromogenic substrates of serine proteinases described herein is straightforward and can be applied to design substrates for other proteases.

Application of specific cell permeable cathepsin G inhibitors resulted in reduced antigen processing in primary dendritic cells.

The serine protease cathepsin G (CatG) is expressed in primary antigen-presenting cells and regulates autoantigen processing in CatG pre-loaded fibroblasts. To further investigate the function of CatG in the major histocompatibility complex (MHC) class II loading compartments, a specific, cell permeable CatG-inhibitor is needed. In this study, several CatG-inhibitors were tested for their ability to penetrate the cell membrane of peripheral blood mononuclear cells (PBMC). We find that the commercially available reversible CatG-specific inhibitor I (CatG inhibitor) and the irreversible Suc-Val-Pro-Phe(P) (OPh)(2) (Suc-VPF) are both cell permeable and specifically inhibit intracellular CatG in the PBMC. Furthermore, selective inhibition of CatG resulted in reduced tetanus toxin C-fragment (TTC) and hemagglutinin (HA) processing and presentation to CD4(+) T cells. We conclude that these CatG inhibitors can be used for both antigen-processing studies and for modulation of T cell response in situ and in vivo.

Peptomeric analogues of trypsin inhibitor SFTI-1 isolated from sunflower seeds

A series of linear and monocyclic analogues of trypsin inhibitor SFTI-1 isolated from sunflower seeds, modified by N-(4-aminobutyl)glycine (Nlys) and N-benzylglycine (Nphe), were obtained by the solid-phase method. Some of these peptomers displayed trypsin or chymotrypsin inhibitory activity. In contradiction to the literature data, in most analogues peptide bonds formed by these peptoid monomers were at least partially hydrolyzed by the experimental enzymes at two different pH (3.5 and 8.3). Nevertheless, the replacement of Phe present in the P(1) substrate specificity of linear inactive SFTI-1 analogue with Nphe, yielded a potent chymotrypsin inhibitor. The introduction of one cyclic element (a disulfide bridge or head-to-tail cyclization) to the analogues synthesized significantly increased their proteinase resistance.

Development of sensitive cathepsin G fluorogenic substrate using combinatorial chemistry methods.

In the current work, a synthesis of new sensitive fluorescence substrates of cathepsin G is reported. The substrate sequence was selected using combinatorial chemistry methods. The starting structure of chromogenic cathepsin G substrate Ac-Phe-Val-Thr-Gnf-ANB-NH(2), where Gnf stands for 4-guanidine-l-phenylalanine, was modified by replacing the acetyl moiety with a residue of 7-methoxycoumarin-4-yl acetic acid (Mca) that served as a fluorescence donor. An amide of amino benzoic acid (ANB-NH(2)) was used as an acceptor. This peptide, exhibiting effective fluorescence resonance energy transfer (FRET) phenomena, was used as a starting structure to construct the library Mca-Phe-Val-Thr-Gnf-X(1)-X(2)-ANB-NH(2), where in both variable X positions all proteinogenic amino acid residues except Cys were introduced. Deconvolution of such a library, performed by the iterative method in solution, revealed prime site preferences of cathepsin G. Finally, the most susceptible sequence, Mca-Phe-Val-Thr-Gnf-Ser-Trp-ANB-NH(2), was selected. The determined value of the specificity constant (k(cat)/K(M) = 252 x 10(3)M(-1)xs(-1)) was two orders of magnitude higher than that obtained for the parent compound. By the use of this substrate, we were able to detect as little as 70 pM of the enzyme studied.

Low-Molecular-Weight Aldehyde Inhibitors of Cathepsin G

A series of aldehyde inhibitors with the general formula Ac-Phe-Val-Thr-X-CHO, where X = Lys, Arg, Phe, Tyr, p-nitro-L-phenylalanine (Nif), p-amino-L-phenylalanine (Amf), p-guanidine-L-phenylalanine (Gnf), pyridyl-L-alanine (Pal), was synthesized. The starting structure of this series based on our previous work on cathepsin G chromogenic substrates. The synthesis of all compounds was performed in solid phase applying Fmoc chemistry. We investigated the inhibitory potency of the obtained compounds against cathepsin G and bovine alpha-chymotrypsin and evaluated their dissociation constants (K(i)). The studied peptides displayed different inhibition profiles and potency. As a result, a potent and selective inhibitor of cathepsin G with the sequence Ac-Phe-Val-Thr-Gnf-CHO, displaying K(i) = 22 nM was obtained.