Evaluation of phosphoramidon and three synthetic phosphonates for inhibition of botulinum neurotoxin B catalytic activity

Three putative metalloprotease inhibitors were synthesized and tested for their ability to inhibit the catalytic activity of botulinum neurotoxin B light chain (BoNT/B LC). The compounds were designed to emulate the naturally occurring metalloprotease inhibitor phosphoramidon, which has been reported to be a weak antagonist of BoNT/B action. All three analogs contained the dipeptide Phe-Glu in place of Leu-Trp of phosphoramidon and possessed a phenyl, ethyl or methyl group in place of the rhamnose sugar of the parent compound. The inhibitors were evaluated in a cell-free assay based on the detection of a fluorescent product following cleavage of a 50-mer synaptobrevin peptide ([Pya(88)] S 39-88) by BoNT/B LC. This peptide corresponds to the hydrophilic core of synaptobrevin-2 and contains a fluorescent analog L-pyrenylalanine (Pya) in place of Tyr(88). Cleavage of [Pya(88)] S 39-88 by BoNT/B LC gives rise to fragments of 38 and 12 amino acid residues. Quantification of BoNT/B-mediated substrate cleavage was achieved by separating the 12-mer fragment (FETSAAKLKRK-Pya) that contains the C-terminal fluorophore and measuring fluorescence at 377 nm. The results indicate that the phenyl-substituted synthetic compound ICD 2821 was slightly more active than phosphoramidon, but analogs with methyl or ethyl substitutions were relatively inactive. These findings suggest that phosphonate monoesters may be useful for providing insights into the structural requirement of BoNT/B protease inhibitors.     

Camelid single domain antibodies (VHHs) as neuronal cell intrabody binding agents and inhibitors of Clostridium botulinum neurotoxin (BoNT) proteases

Botulinum neurotoxins (BoNTs) function by delivering a protease to neuronal cells that cleave SNARE proteins and inactivate neurotransmitter exocytosis. Small (14 kDa) binding domains specific for the protease of BoNT serotypes A or B were selected from libraries of heavy chain only antibody domains (VHHs or nanobodies) cloned from immunized alpacas. Several VHHs bind the BoNT proteases with high affinity (K_D near 1 nM) and include potent inhibitors of BoNT/A protease activity (K_i near 1 nM). The VHHs retain their binding specificity and inhibitory functions when expressed within mammalian neuronal cells as intrabodies. A VHH inhibitor of BoNT/A protease was able to protect neuronal cell SNAP25 protein from cleavage following intoxication with BoNT/A holotoxin. These results demonstrate that VHH domains have potential as components of therapeutic agents for reversal of botulism intoxication.     

A Highly Specific Monoclonal Antibody for Botulinum Neurotoxin Type A-Cleaved SNAP25

Botulinum neurotoxin type-A (BoNT/A), as onabotulinumtoxinA, is approved globally for 11 major therapeutic and cosmetic indications. While the mechanism of action for BoNT/A at the presynaptic nerve terminal has been established, questions remain regarding intracellular trafficking patterns and overall fate of the toxin. Resolving these questions partly depends on the ability to detect BoNT/A’s location, distribution, and movement within a cell. Due to BoNT/A’s high potency and extremely low concentrations within neurons, an alternative approach has been employed. This involves utilizing specific antibodies against the BoNT/A-cleaved SNAP25 substrate (SNAP25₁₉₇) to track the enzymatic activity of toxin within cells. Using our highly specific mouse monoclonal antibody (mAb) against SNAP25₁₉₇, we generated human and murine recombinant versions (rMAb) using specific backbone immunoglobulins. In this study, we validated the specificity of our anti-SNAP25₁₉₇ rMAbs in several different assays and performed side-by-side comparisons to commercially-available and in-house antibodies against SNAP25. Our rMAbs were highly specific for SNAP25₁₉₇ in all assays and on several different BoNT/A-treated tissues, showing no cross-reactivity with full-length SNAP25. This was not the case with other reportedly SNAP25₁₉₇-selective antibodies, which were selective in some, but not all assays. The rMAbs described herein represent effective new tools for detecting BoNT/A activity within cells.     

Tandem fluorescent proteins as enhanced FRET-based substrates for botulinum neurotoxin activity

The light chain of botulinum neurotoxin A (BoNT/A-LC) is a zinc-metalloprotease that requires two extended exosites for optimal substrate binding and recognition of its intracellular target SNAP25. CFP and YFP connected through SNAP25 peptide (141-206) containing both exosites (CsY) has been used in a FRET-based assay for BoNT/A. To further improve the FRET efficiency in this BoNT/A substrate for in vitro high-throughput assays, we explored the feasibility of enhancing the capture of CFP emission by doubling the number of YFP acceptors. In comparison to CsY, the tandem fluorescence substrates CsYY and YsCsY enhanced the ratiometric fluorescence signal between YFP and CFP. YsCsY, containing two substrate sites, offered the greatest fluorometric change upon toxin-catalyzed cleavage. In addition to known approaches for enhancing fluorescence yield through various mutations, this alternative tandem substrate approach can boost the FRET signal and is particularly useful for substrates requiring extensive exosite recognition for specificity.     

Exploring the stereochemical requirements for protease inhibition by ureidopeptides

Abstract: A novel ‘ureidopeptide’ substrate analog inhibitor of the HIV‐1 protease, created by substitution of a urea for the scissile amide bond of a hexapeptide substrate, was synthesized and tested for inhibition of HIV‐1 protease. This inhibitor was designed as a stereochemical mutant of an earlier ureidopeptide inhibitor in which the P1′ phenylalanine residue was changed from an l ‐isomer to a d ‐isomer. This was done in an attempt to increase binding to the enzyme by compensating for a lengthening of the peptide backbone. The inhibitor was synthesized from two protected tripeptide precursors using an oxidative Hoffmann rearrangement of a C‐terminal peptide amide. The new inhibitor was found to inhibit HIV‐1 protease with an observed IC₅₀ of 47 μm .     

Buforin I, a natural peptide, inhibits botulinum neurotoxin B activity in vitro

Botulinum neurotoxin B (BoNT/B) serotype specifically cleaves between the amino acids glutamine and phenylalanine (Q and F bond) in position 76-77 of synaptobrevin (VAMP2). We evaluated peptides that contain the QF cleavage site but are not identical in primary structure to the VAMP2 sequence surrounding the QF site for both inhibition of BoNT/B proteolytic activity and as substrates for BoNT/B. A reverse-phase high-performance liquid chromatography (RP-HPLC) method was used to measure digested peptides. A dose as high as 600 microM of substance P, and 11-amino acid peptide containing the QF bond, was neither a substrate nor inhibitor of BoNT/B in our assay, suggesting that more than the QF bond is required to be recognized by BoNT/B. Buforin I (B-I, QF site 24-25) is 39 amino acids in length, and sequence comparison of B-I and VAMP2 indicated a similarity of 18% for conserved amino acids around the QF site. Furthermore, computer-aided secondary structure computations predict alpha-helical structures flanking the QF site for VAMP2 and for the upstream sequence of B-I. Although predictions for the downstream sequence give nearly equal tendencies for alpha-helical and beta-sheet structures, Yi et al. showed that the downstream sequence is likely to be the alpha-helix based on their examination of buforin II (B-II, a 21-amino acid subset of B-I (16-36)), which includes the QF site and the downstream sequence of B-I. Buforin I was found not to be a substrate for BoNT/B; however, B-I dose dependently and competitively inhibited BoNT/B activity, yielding IC(50) = 1 x 10(-6) M. In contrast, B-II was not a substrate for BoNT/B and exhibited only 25% of the B-I inhibition of BoNT/B. Two additional B-I deletion peptides were tested for inhibition of BoNT/B proteolysis: peptide 36 (36 mer; containing B-I amino acids 1-36) and peptide 24 (24 mer; B-I amino acids 16-39). Peptide 24 had a similar inhibitory effect to B-II (ca. 25% of B-I) but peptide 36 was almost 50% as potent as B-I. These findings suggest that the buforin tertiary structure is important for the inhibitory activity of these peptides for BoNT/B.     

Combined solid‐phase/solution synthesis of large ribonuclease A C‐terminal peptides containing a non‐natural proline analog*

Abstract: Three large peptides corresponding to the 65–124 (60‐mer), 72–124 (53‐mer), and 77–124 (48‐mer) sequence of bovine pancreatic ribonuclease A (RNase A) were assembled from either two or three shorter protected peptide fragments by chemical coupling in solution. The fragments were synthesized manually by 9‐fluorenylmethyloxycarbonyl (Fmoc)‐based solid‐phase peptide chemistry in plastic syringes, and subsequently purified by normal‐phase high‐performance liquid chromatography on a silica gel column. The main aim of this work was to incorporate sterically hindered l ‐5,5‐dimethylproline (dmP) as a substitute for Pro⁹³ into the sequence of RNase A in order to constrain the –Tyr⁹²‐Pro⁹³– peptide group to a single cis‐conformation.     

Protease‐catalyzed fragment condensation via substrate mimetic strategy: a useful combination of solid‐phase peptide synthesis with enzymatic methods

Abstract: The concept of substrate mimetic strategy represents a new powerful method in the field of enzymatic peptide synthesis. This strategy takes advantage of the shift in thesite‐specific amino acid moiety from the acyl residue to the ester‐leaving group of the carboxyl component enabling acylation of the enzyme by nonspecific acyl residues. As a result, peptide bond formation occurs independently of the primary specificity of proteases. Moreover, because of the coupling of nonspecific acyl residues, the newly formed peptide bond is not subject to secondary hydrolysis achieving irreversible peptide synthesis. Here, we report the combination of solid‐phase peptide synthesis with substrate mimetic‐mediated enzymatic peptide fragment condensations. First, the utility of the oxime resin strategy for the synthesis of peptide fragments in the form of substrate mimetics esterified as 4‐guanidinophenyl‐, phenyl‐ and mercaptopropionic acid esters was investigated. The study was completed by using the resulting N^α‐protected peptide esters as acyl donors in trypsin‐, α‐chymotrypsin‐ and V8 protease‐catalyzed fragment condensations.     

Synthesis of dermorphin‐(1–4) derivatives catalyzed by proteases in organic solvents*

Abstract: In order to extend the use of proteases to organic synthesis and seek the rules of enzymatic reactions in organic media, we focused on unnatural substrates for proteases to form amide bonds. In this paper, the study of unnatural substrates containing d ‐amino acid residue, which act as acyl acceptors as well as acyl donors for proteases in organic media, is reported. Dermorphin is a heptapeptide (H‐Tyr‐d ‐Ala‐Phe‐Gly‐Tyr‐Pro‐Ser‐NH₂) with potent analgesic activity. The N‐terminal tetrapeptide is the minimum sequence that retains dermorphin activity, and is selected as the model compound in our study. Two dermorphin‐(1–4) derivatives, Boc‐Tyr‐d ‐Ala‐Phe‐Gly‐N₂H₂Ph and Boc‐Tyr‐d ‐Ala‐Phe‐Gly‐NH₂, which contained a d ‐amino acid residue, were synthesized by proteases in organic media for the first time. The synthesis of these two dermorphin‐(1–4) derivatives could be catalyzed by subtilisin with Boc‐Tyr‐d ‐Ala‐OCH₂CF₃ as an acyl donor substrate in AcOEt. The synthesis of dermorphin‐(1–2) derivative Boc‐Tyr‐d ‐Ala‐N₂H₂Ph was catalyzed by α‐chymotrypsin in different organic solvents and d ‐Ala‐N₂H₂Ph was used as an acyl acceptor substrate. Factors influencing the above enzymatic reactions were systematically studied.     

Facile synthesis of orthogonally protected amino acid building blocks for combinatorial N‐backbone cyclic peptide chemistry

Abstract: Protected N^α‐(aminoallyloxycarbonyl) and N^α‐(carboxyallyl) derivatives of all natural amino acids (except proline), and their chiral inverters, were synthesized using facile and efficient methods and were then used in the synthesis of N^α‐backbone cyclic peptides. Synthetic pathways for the preparation of the amino acid building units included alkylation, reductive amination and Michael addition using alkylhalides, aldehydes and α,β‐unsaturated carbonyl compounds, and the corresponding amino acids. The resulting amino acid prounits were then subjected to Fmoc protection affording optically pure amino acid building units. The appropriate synthetic pathway for each amino acid was chosen according to the nature of the side‐chain, resulting in fully orthogonal trifunctional building units for the solid‐phase peptide synthesis of small cyclic analogs of peptide loops (SCAPLs^?). N^α‐amino groups of building units were protected by Fmoc, functional side‐chains were protected by t‐Bu/Boc/Trt and N‐alkylamino or N‐alkylcarboxyl were protected by Alloc or Allyl, respectively. This facile method allows easy production of a large variety of amino acid building units in a short time, and is successfully employed in combinatorial chemistry as well as in large‐scale solid‐phase peptide synthesis. These building units have significant advantage in the synthesis of peptido‐related drugs.     

Facile degradative lactonization of Gln‐Arg and Gln‐Phe hydroxyethylene dipeptide derivatives*

Abstract: We have found that hydroxyethylene (HE) dipeptide analogs of Gln‐Arg and Gln‐Phe are unusually susceptible to acid catalyzed lactonization. The synthesis of substrate‐based transition state analog inhibitors of botulinum neurotoxin metalloprotease inhibitors that contain the Gln‐Arg or the Gln‐Phe HE units is complicated by this facile degradative lactonization.     

Synthesis and study of normal,enantio, retro,and retroenantio isomers of cecropin A-melittin hybrids,their end group effects and selective enzyme inactivation

In our effort to understand the structural requirements for the antimicrobial activity of cecropin A (CA) and melittin (M), we synthesized the normal, enantio, retro and retroenantio hybrid analogs; we related activity to their sequence, chirality, amide bond direction (helix dipole) and end group charges. To compare the effect of the end groups, each of these analogs was synthesized both with an acid and an amide C-terminus and also with and without an N^α-acetyl N-terminus. The all-l - and all-d -enantiomers of several cecropin-melittin hybrids were previously found to be equally potent against several bacterial species, and no chiral effect was observed. This general rule has now been confirmed and extended. However, two exceptions have been found. All-l -CA(1-13)M(1-13) acid was 5 times and 9 times less potent than the all-d -analog, respectively, toward Gram-positive Staphylococcus aureus and Gram-negative Pseudomonas aeruginosa. A11-l -CA(1-7)M(2-9) acid was 5 times and 14 times less active against S. aureus and P. aeruginosa, respectively, than its all-d acid isomer. The corresponding d - and l -retro analogs differed only marginally. A role for proteolytic enzymes has been implicated as a cause for these differences in the activities of l - and d -enantiomers. In all cases, blocking the α-amine by acetylation had no significant effect on potency. The retro and retroenantio analogs of CA(1-13)M(1-13) acid were as potent as their normal and enantio analogs against all the test bacteria. The C-terminal amides also showed similar potency against four test bacteria. It should be noted that the negative end of the helix dipole of a normal peptide points toward the C-terminus, whereas it points away in the case of a retro derivative when viewed in the direction of the normal sequence.     

Synthesis of side-chain to side-chain cyclized peptide analogs on solid supports

Cyclic peptide structures of the type -Lys-R₁—R_n-Glu- can be synthesized on the Merrifield resin by assembling the peptide chain using Nα-Fmoc-amino acids and Boc and tert.-butyl protection for the side-chains of Lys and Glu, respectively. If residues R₁ to R_n contain side-chain functional groups, TFA-resistant protection is required. After TFA treatment cyclization on the resin can be performed with appropriate coupling reagents. The formation of such cyclic structures may be preceded or followed by peptide chain assembly using Nα-Boc-amino acids and the entire peptide chain containing the cyclic portion is finally cleaved by HF treatment. Using this principle we synthesized the following opioid peptide related cyclic analogs: H-Tyr-d -Lys-Gly-Phe-Glu-NH₂ (I), H-Tyr-Lys-Gly-Phe-Glu-NH₂ (II), H-Tyr-d -Lys-Phe-Glu-NH₂ (III), H-Tyr-d -Glu-Gly-Phe-Lys-NH₂ (IV), H-Tyr-d -Glu-Phe-Lys-NH₂ (V), H-Tyr-d -Orn-Gly-Glu-NH₂ (VI) and H-Tyr-d -Ala-Lys-Phe-Glu-NH₂ (VII). Cyclic monomers were obtained in all cases, as demonstrated by mass spectrometry. Analysis of side-products revealed a slow-down of the HF deprotection of O-benzylated tyrosine as a consequence of hydrophobic interactions as well as the formation of a side-chain-linked antiparallel cyclic dimer in the case of compound VI. In conclusion, the described method permits the convenient preparation of peptide analogs cyclized via amide bond formation between side-chain amino and carboxyl groups in reasonable yield.     

New peptidic cysteine protease inhibitors derived from the electrophilic alpha-amino acid aziridine-2,3-dicarboxylic acid

Three different types of peptides containing aziridine-2, 3-dicarboxylic acid (Azi) as an electrophilic alpha-amino acid at different positions within the peptide chain (type I, N-acylated aziridines with Azi as C-terminal amino acid; type II, N-unsubstituted aziridines with Azi as N-terminal amino acid; type III, N-acylated bispeptidyl derivatives of Azi) have been synthesized and tested as inhibitors of the cysteine proteases papain, cathepsins B, L, and H, and calpains I and II, as well as against several serine proteases, one aspartate, and one metalloprotease. All aziridinyl peptides are specific cysteine protease inhibitors. Papain and cathepsins B and L are inhibited irreversibly, whereas cathepsin H and calpains are inhibited in a non-time-dependent manner. Some compounds turned out to be substrates for serine proteases and for the metalloprotease thermolysin. Remarkable differences can be observed between the three different types of inhibitors concerning stereospecificity, pH dependency of inhibition, selectivity between different cysteine proteases, and the importance of a free carboxylic acid function at the aziridine ring for inhibition. Above all type II inhibitors, aza analogues of the well-known epoxysuccinyl peptides, are potent cysteine protease inhibitors. With the exception of BOC-Leu-Gly-(S, S+R,R)-Azi-(OEt)2 (28a+b), a highly selective and potent cathepsin L inhibitor, N-acylated aziridines of type I are weaker inhibitors than type II or type III compounds. The observed results can be explained by different binding modes of the three types of inhibitors with respect to their orientation in the S- and S'-binding sites of the enzymes. Furthermore, the presence of a protonated aziridine N modifies the binding mode of type II inhibitors.     

Synthesis of peptide p-nitroanilides mimicking fibrinogen- and hirudin-binding to thrombin Design of slow reacting thrombin substrates

The design and synthesis of 20 peptide p-nitroanilides is described. The nitroanilides are used as thrombin substrates that share uncommon properties. These substrates are tested for their applicability to measure thrombin generation in activated plasma. This technique requires substrates that must slowly but selectively be hydrolyzed by thrombin. To ensure selectivity, thrombin's natural substrate and its most potent inhibitor were used as lead compounds. Eighteen peptide p-nitroanilides were synthesized using human fibrinogen Aα[7-16] decapeptide as lead structure since this fragment constitutes the minimal sequence which binds to thrombin with high affinity. Two other chromogenic substrates were designed using 5-amino-2-nitrobenzoic acid as the chromophore. A peptide giving subsite interactions with the fibrinogen recognition exosite was coupled to the carboxylic function. As the peptide segment, the carboxyl terminal part of hirudin (residues 50-65) was chosen to ensure highly specific thrombin recognition. From the 20 nitroanilides synthesized. We indeed obtained a number of compounds which can be used as substrate in the thrombin generation assay. © Munksgaard 1996.     

A solid‐phase synthetic strategy for the preparation of peptide‐based affinity labels: synthesis of dynorphin A analogs

Abstract: Solid‐phase synthetic methodology was developed for the preparation of peptide‐based affinity labels. The initial peptides synthesized were dynorphin A (Dyn A) analogs [Phe(p‐X)⁴,d ‐Pro¹⁰]Dyn A(1–11)NH₂ containing isothiocyanate (X = –N=C=S) and bromoacetamide (X = –NHCOCH₂Br) groups. The peptides were assembled on solid supports using Fmoc‐protected amino acids, and the side chain amine to be functionalized, Phe(p‐NH₂), was protected by the Alloc (allyloxycarbonyl) group. Following removal of the Alloc group by palladium(0), the reactive isothiocyanate and bromoacetamide functionalities were successfully introduced while the peptides were still attached to the resin. Synthesis of these peptides was carried out on polystyrene (PS) and polyethylene glycol–polystyrene (PEG–PS) resins containing the PAL [peptide amide linker, 5‐(4‐Fmoc‐aminomethyl‐3,5‐dimethoxyphenoxy)valeric acid] linker. Both the rate of Alloc deprotection and the purity of the crude affinity‐labeled peptides obtained were found to be dependent on the resin used for peptide assembly.     

Synthesis and Anti‐Inflammatory Evaluation of Novel C66 Analogs for the Treatment of LPS‐Induced Acute Lung Injury

We previously reported a symmetric monocarbonyl analog of curcumin (MACs), C66, which demonstrated potential anti‐inflammatory activity and low toxicity. In continuation of our ongoing research, we designed and synthesized 34 asymmetric MACs based on C66 as a lead molecule. A majority of the C66 analogs effectively inhibited LPS induction of TNF‐α and IL‐6 expression. Additionally, a preliminary SAR was conducted. Furthermore, active compounds 4a11 and 4a16 were found to effectively reduce the W/D ratio in the lungs and the protein concentration in the bronchoalveolar lavage fluid (BALF). Meanwhile, a histopathological examination indicated that these two analogs significantly attenuate tissue injury in the lungs with LPS‐induced ALI rats. 4a11 and 4a16 also inhibited mRNA expression of several inflammatory cytokines, including TNF‐α, IL‐6, IL‐1β, COX‐2, ICAM‐1 and VCAM‐1, in the Beas‐2B cells after LPS challenge. Altogether, the data exhibit a series of new C66 analogs as promising anti‐inflammatory agents for the treatment of LPS‐induced ALI.     

[Ala12]MCD peptide: a lead peptide to inhibitors of immunoglobulin E binding to mast cell receptors1

Abstract: An effort was made to discover mast cell degranulating (MCD) peptide analogs that bind with high affinity to mast cell receptors without triggering secretion of histamine or other mediators of the allergic reaction initiated by immunoglobulin E (IgE) after mast cell activation. Such compounds could serve as inhibitors of IgE binding to mast cell receptors. An alanine scan of MCD peptide reported previously showed that the analog [Ala¹²]MCD was 120‐fold less potent in histamine‐releasing activity and fivefold more potent in binding affinity to mast cell receptors than the parent MCD peptide. Because this analog showed marginal intrinsic activity and good binding affinity it was subsequently tested in the present study as an IgE inhibitor. In contrast to MCD peptide, [Ala¹²]MCD showed a 50% inhibition of IgE binding to the FcεRIα mast cell receptor by using rat basophilic leukemia (RBL‐2H3) mast cells and fluorescence polarization. Furthermore, in a β‐hexosaminidase secretory assay, the peptide also showed a 50% inhibition of the secretion of this enzyme caused by IgE. An attempt was made to relate structural changes and biologic differences between the [Ala¹²]MCD analog and the parent MCD peptide. The present results show that [Ala¹²]MCD may provide a base for designing agents to prevent IgE/FcεRIα interactions and, consequently, allergic conditions.     

Synthesis and biological evaluation of isosteric analogs of mandipropamid for the control of oomycete pathogens

A series of isosteric analogs of mandipropamid were designed and synthesized via ‘click chemistry’. The amide bond of mandipropamid was substituted by a 1,2,3‐triazole functional group. The bioassay results have indicated that some of the title compounds exhibited moderate fungicidal activity against Pseudoperonospora cubensis, and the activity has been systematically studied as a function of molecular structure. The low activity of the mandipropamid analog that contains a lipid chain is likely due to the presence of a weak hydrogen bond donor in the 1,2,3‐triazole. Furthermore, we have performed the molecular modeling and found that N‐methylamide could be more effective than amide as the surrogates to 1,2,3‐triazole, which ultimately leads to a longer distance (1.1 Å longer) between the two substitutes in the 1,4‐disubstituted 1,2,3‐triazole compound.     

Analogs of oxytocin containing a pseudopeptide Leu-Gly bond of cis and trans configuration*

Analogs of deamino-oxytocin wherein the Leu-Gly peptide bond has been replaced by a tetrazole moiety or by a double bond of trans configuration were synthesized and their biological activities evaluated. Trans double bond was found to be the most appropriate substitution for the amide bond (uterotonic activity 24% of the deamino-oxytocin). In the case of all three analogs low but prolonged galactogogic activity was found and the ratio of uterotonic in vitro and in vivo activity was surprisingly high (ranging from 4.5 to 20).