Synthesis of boronic acid analogues of alpha-amino acids by introducing side chains as electrophiles.

A synthetic route has been developed which has allowed us to prepare novel alpha-aminoboronic acids as inhibitors of serine proteases. These compounds were prepared to study the roles of proteases in biological systems. This methodology affords alpha-aminoboronic acids with the general formula R'-NHCH(R)BO(2)-pinanediol, where R = -CH(2)CHF(2), -CH(2)CO(2)tBu, and -(CH(2))(2)CO(2)Me and R' = either H or C(O)R". The latter two compounds are the boronic acid analogues of the natural amino acids aspartic acid and glutamic acid with the side chain carboxylate protected as a tert-butyl or a methyl ester, respectively. Following acylation of the amino group, the side chain tert-butyl ester of boroaspartic acid was removed by treatment with TFA. Boroglutamic acid was obtained as the free boronic acid by hydrolysis with HCl. Prior syntheses of alpha-aminoboronic acids involve the initial addition of an organometallic reagent to a trialkyl borate ester. These conditions do not allow the preparation of compounds with functionalities that are not stable to the strongly basic reaction conditions. The methodology described here allows the preparation of alpha-aminoboronic acids by introducing side chains as electrophiles. This is particularly advantageous for side chains which are prone to elimination or unwanted enolate formation. Specifically, BrCH(2)CHF(2), BrCH(2)COO(t)Bu, and CH(2)=CHCOOMe were allowed to react with the stabilized anion of (phenylthio)methane boronate, PhSCH(2)BO(2)C(6)H(12), to give the substituted boronate. The substituted (phenylthio)methane boronate was converted to the corresponding sulfonium ion by treatment with methyl iodide and subsequently displaced with iodide. The alpha-iodo derivative was converted to the amine by conventional methods.

Inhibition of the bovine viral diarrhoea virus NS3 serine protease by a boron-modified peptidyl mimetic of its natural substrate.

Bovine viral diarrhoea virus (BVDV) is closely related to hepatitis C virus (HCV), and has been used as a surrogate virus in drug development for HCV infection. Similar to HCV, BVDV-encoded NS3 serine proteinase is responsible for multiple cleavages in the viral polyprotein, generating mature NS4A, NS4B, NS5A and NS5B proteins. NS3-dependent cleavage sites of BVDV contain a strictly conserved leucine at P1, and either serine or alanine at P1'. The full length BVDV NS3/4A serine protease has been cloned and expressed in bacterial cells. The enzyme has been purified from the soluble portion of Escherichia coli via a two-step purification procedure employing chromatography on heparin resin and gel filtration. The protease activity was characterized using in vitro translated BVDV NS4A/B and NS5A/B polyprotein substrates. A boronic acid analogue of the BVDV NS4A/NS4B cleavage site was synthesized and shown to be an efficient inhibitor of the NS3 serine protease in vitro. The compound, designated DPC-AB9144-00, inhibited approximately 75% of the NS3/4 activity at 10 microM with the NS4A/B substrate. However, no antiviral activity was detected with DPC-AB9144-00 in BVDV-infected Madin-Darby bovine kidney cells at concentrations as great as 90 pM, suggesting permeability or that other cellular-derived limitations were present.

Drift ratchet

We consider a silicon wafer, pierced by millions of identical pores with periodically varying diameters but without spatial inversion symmetry (ratchet profile). When a liquid is periodically pumped back and forth through the pores, our theory predicts a net transport of suspended micrometer-sized particles (drift ratchet). The direction of this particle current depends very sensitively on the size of the particles. For typical parameter values of the experiment, two different types of particles at an initially homogeneous 1:1 mixture are spatially separated with a purity beyond 1:1000 on a time scale of a few hours in comparably large quantities. This result is due to the highly parallel architecture of the device. The experimental realization of the setup, presently under construction, thus appears to be a promising new particle separation device, possibly superior to existing methods for particles sizes on the micrometer scale.

7-Azabicycloheptane carboxylic acid: a proline replacement in a boroarginine thrombin inhibitor.

[formula: see text] The synthesis of thrombin inhibitor 3, which incorporates conformationally constrained 7-azabicycloheptane carboxylic acid (1) as a proline replacement, is described. The inhibition constant (Ki(thrombin) = 2.9 nM) indicates that 1 is a reasonable replacement of proline in the formation of a beta-turn tripeptide mimetic.

Thrombin and human plasma kallikrein inhibition during simulated extracorporeal circulation block platelet and neutrophil activation.

Cardiopulmonary bypass causes hemorrhagic complications, and initiates a chemical and cellular inflammatory response. Contact of blood with synthetic surfaces leads to qualitative and quantitative alterations in platelets, neutrophils, complement, and contact systems. Despite the fact that cardiopulmonary bypass is carried out in the presence of high doses of heparin, there is significant activation of both platelets and neutrophils. Thrombin is protected on cell and fibrin surfaces from antithrombin, even in the presence of high doses of heparin (approximately 5 U/ml). We therefore studied the effect of a small (Mr = 497), highly effective (Ki = 41 pM), reversible tripeptide inhibitor of thrombin, DUP 714 (1 microM), in a well characterized model of simulated extracorporeal circulation. In the absence of DUP 714, platelet counts decreased by 75% 5 min after the start of extracorporeal bypass and increased to 48% at 120 min of recirculation. DUP 714 significantly preserved platelet counts, decreased plasma levels of platelet beta-thromboglobulin levels, but did not prevent a decrease in sensitivity of platelets to adenosine diphosphate. Kallikrein-C1-inhibitor and C1-C1-inhibitor complexes increased progressively from 0.32 U/ml to 0.67 U/ml and from 4.45 U/ml to 7.25 U/ml, respectively, during 120 min of recirculation without DUP 714. Addition of DUP 714 significantly inhibited kallikrein-C1-inhibitor complex formation but did not affect C1-C1-inhibitor complexes. In the absence of DUP 714, human neutrophil elastase levels rose from a baseline of 0.01 +/- 0.00 microg/ml to 1.18 +/- 0.21 microg/ml during 120 min of recirculation. Human neutrophil elastase release at 120 min was significantly inhibited in the presence of DUP 714 to 37% of the value with heparin alone. These results indicated that addition of this novel thrombin (and kallikrein) inhibitor to heparin preserved platelet counts, decreased platelet secretion, and provided the additional benefit of partially blocking neutrophil activation during simulated extracorporeal circulation.

Electrophysiology in the eukaryotic model cell Saccharomyces cerevisiae.

Since the mid-1980s, use of the budding yeast, Saccharomyces cerevisiae, for expression of heterologous (foreign) genes and proteins has burgeoned for several major purposes, including facile genetic manipulation, large-scale production of specific proteins, and preliminary functional analysis. Expression of heterologous membrane proteins in yeast has not kept pace with expression of cytoplasmic proteins for two principal reasons: (1) although plant and fungal proteins express and function easily in yeast membranes, animal proteins do not, at least yet; and (2) the yeast plasma membrane is generally regarded as a difficult system to which to apply the standard electrophysiological techniques for detailed functional analysis of membrane proteins. Especially now, since completion of the genome-sequencing project for Saccharomyces, yeast membranes themselves can be seen as an ample source of diverse membrane proteins - including ion channels, pumps, and cotransporters - which lend themselves to electrophysiological analysis, and specifically to patch-clamping. Using some of these native proteins for assay, we report systematic methods to prepare both the yeast plasma membrane and the yeast vacuolar membrane (tonoplast) for patch-clamp experiments. We also describe optimized ambient conditions - such as electrode preparation, buffer solutions, and time regimens - which facilitate efficient patch recording from Saccharomyces membranes. There are two main keys to successful patch-clamping with Saccharomyces. The first is patience; the second is scrupulous cleanliness. Large cells, such as provided by polyploid strains, are also useful in yeast patch recording, especially while the skill required for gigaseal formation is being learned. Cleanliness is aided by (1) osmotic extrusion of protoplasts, after minimal digestion of yeast walls; (2) use of a rather spare suspension of protoplasts in the recording chamber; (3) maintenance of continuous chamber perfusion prior to formation of gigaseals; (4) preparation (pulling and filling) of patch pipettes immediately before use; (5) application of a modest pressure head to the pipette-filling solution before the tip enters the recording bath; (6) optical control for debris at the pipette tip; and (7) discarding of any pipette that does not "work" on the first try at gigaseal formation. Other useful tricks toward gigaseal formation include the making of protoplasts from cells grown aerobically, rather than anaerobically; use of sustained but gentle suction, rather than hard suction; and manipulation of bath temperature and/or osmotic strength. Yeast plasma membranes form gigaseals with difficulty, but these tend to be very stable and allow for long-term cell-attached or whole-cell recording. Yeast tonoplasts form gigaseals with ease, but these tend to be unstable and rarely allow recording for more than 15 min. The difference of stability accrues mainly because of the fact that yeast protoplasts adhere only lightly to the recording chamber and can therefore be lifted away on the patch pipette, whereas yeast vacuoles adhere firmly to the chamber bottom and are subsequently stressed by very slight relative movements of the pipette. With plasma membranes, conversion from cell-attached recording geometry to isolated ISO patch (inside-out) geometry is accomplished by blowing a fine stream of air bubbles across the pipette tip; to whole-cell recording geometry, by combining suction and one high-voltage pulse; and from whole-cell to OSO patch (outside-out) geometry, by sudden acceleration of the bath perfusion stream. With tonoplasts, conversion from the vacuole-attached recording geometry to whole-vacuole geometry is accomplished by application of a large brief voltage pulse; and further conversion to the OSO patch geometry is carried out conventionally, by slow withdrawal of the patch pipette from the vacuole, which usually remains attached to the chamber bottom.

Physiological characterization of the yeast plasma membrane outward rectifying K+ channel, DUK1 (TOK1), in situ.

The major voltage-dependent ion channel in the plasma membrane of Saccharomyces cerevisiae, a conspicuous outwardly rectifying K+ channel, was first dubbed YPK1 and later renamed according to its registered gene names (DUK1, TOK1). It has proven novel in both structure and function. Whole-cell patch-clamp studies of the channel directly on yeast protoplasts now extend our earlier description obtained from isolated patches of yeast membrane (Bertl & Slayman, 1992; Bertl et al., 1993), and provide new data both on the contributions of channel properties to yeast physiology and on possible contributions of molecular structure of channel properties. Three recording tactics produce completely equivalent results and thereby allow great flexibility in the design of experiments: whole-cell voltage clamp with sustained voltage steps (approximately 2.5 sec), whole-cell voltage clamp with slow voltage ramps (5 sec, -40 to +100 mV), and time-averaging of single-channel currents. Activation of Duk1 channels under steady-state conditions is dependent upon ATP in the cytoplasmic solution, and the absence of ATP results in channel "rundown"--decreasing numbers of activable channels--over periods of 10 min to 1 hr from the start of patch recording. Several putative serine- and threonine-phosphorylation sites, as well as a variant ATP-binding fold, exist in the molecule as potential mediators of the ATP effects. The channel runs down similarly following cytoplasmic acidification, but is almost completely insensitive to extracellular pH changes (8.0 to 5.5 tested). This remarkable asymmetry may depend on the protein's strongly asymmetric distribution of histidine residues, with 10 out of 12 predicted to lie close to the membrane-cytoplasm interface. Further data confirm the well-recognized observation that changes of K+ concentration, intracellular or extracellular, can shift the gating voltage of Duk1p in the direction of EK. Among the other alkali-metal cations tested, extracellular Rb+ and Cs(+)--but not Na(+)--substitute almost completely for K+. Extracellular TEA+ inhibits whole-cell K+ currents through Duk1p with a KI of 2.8 mM, and does so probably by reducing the single-channel current.

Specific inhibition of plasma kallikrein modulates chronic granulomatous intestinal and systemic inflammation in genetically susceptible rats.

The kallikrein-kinin (K-K) (contact) system is activated during acute and chronic relapsing phases of enterocolitis induced in genetically susceptible Lewis rats by intramural injection of peptidoglycan-polysaccharide (PG-APS). Using the selective plasma kallikrein inhibitor P8720, we investigate whether activation of the K-K system plays a primary role in chronic granulomatous intestinal and systemic inflammation in this model. Group I (negative control) received human serum albumin intramurally. Group II (treatment) received PG-APS intramurally and P8720 orally. Group III (positive control) received PG-APS intramurally and albumin orally. P8720 attenuated the consumption of the contact proteins, high molecular weight kininogen (P<0.03), and factor XI (P<0.04) in group II vs. group III. P8720 decreased chronic intestinal inflammation measured by blinded gross (P<0.01) and histologic (P<0.0005) scores as well as systemic complications (arthritis, splenomegaly, hepatomegaly, leukocytosis, and acute-phase reaction) (P<0.01) in group II as compared with group III. We conclude that relapsing chronic enterocolitis and systemic complications are in part due to plasma K-K system activation, and that inhibition of this pathway is a potential therapeutic approach to human inflammatory bowel disease and associated extraintestinal manifestations.

Biochemical and crystallographic characterization of homologous non-peptidic thrombin inhibitors having alternate binding modes.

The X-ray crystallographic structure of [N-(3-phenylpropionyl)-N-(phenethyl)]-Gly-boroLys-OH (HPBK, Ki = 0. 42 nM, crystallographic R factor to 1.8 A resolution, 19.6%) complexed with human alpha-thrombin shows that the boron adopts a tetrahedral geometry and is covalently bonded to the active serine, Ser195. The HPBK phenethyl aromatic ring forms an edge-to-face interaction with the indole side chain of Trp215. Four HPBK analogs containing either electron-withdrawing or electron-donating substitutents at the 3' position of the phenethyl ring were synthesized in an attempt to modulate ligand affinity by inductive stabilization of the edge-to-face interaction. Refined crystallographic structures of the trifluoromethyl (Ki = 0.37 nM, crystallographic R factor to 2.0 A resolution = 18.7%), fluoro (Ki = 0.60; R factor to 2.3 A resolution = 18.4%), methoxy (Ki = 0.91 nM, R factor to 2.2 A resolution = 19.8%) and methyl (Ki = 0.20 nM, R factor to 2.5 A resolution = 16.9%) HPBK analogs complexed with thrombin revealed two binding modes for the closely related compounds. A less than 1.5-fold variation in affinity was observed for analogs (trifluoromethyl-HPBK and fluoro-HPBK) binding with the edge-to-face interaction. The slight inductive modulation is consistent with the overall weak nature of the edge-to-face interaction. Owing to an unexpected rotation of the phenethyl aromatic ring, the 3' substituent of two analogs, methoxy-HPBK and methyl-HPBK, made direct contact with the Trp215 indole side chain. Increased affinity of the 3' methyl analog is attributed to favorable interactions between the methyl group and the Trp215 indole ring. Differences in inhibitor, thrombin and solvent structure are discussed in detail. These results demonstrate the subtle interplay of weak forces that determine the equilibrium binding orientation of inhibitor, solvent and protein.

Pyrazoles, 1,2,4-triazoles, and tetrazoles as surrogates for cis-amide bonds in boronate ester thrombin inhibitors.

Substituted pyrazoles, 1,2,4-triazoles, and tetrazoles are good surrogates for cis-amide bonds in a series of boronate ester thrombin inhibitors.

Rational design of boropeptide thrombin inhibitors: beta, beta-dialkyl-phenethylglycine P2 analogs of DuP 714 with greater selectivity over complement factor I and an improved safety profile.

The potent boropeptide thrombin inhibitor DuP 714 caused side effects in laboratory animals that appear to be related to its ability to inhibit complement factor I, thereby activating the complement cascade. Using X-ray crystal structure information, we have designed compounds that have greater selectivity for thrombin over factor I and that have reduced tendency to produce these side effects.

Low barrier hydrogen bond is absent in the catalytic triads in the ground state but Is present in a transition-state complex in the prolyl oligopeptidase family of serine proteases.

High frequency proton NMR spectra for two members of the prolyl oligopeptidase class of serine proteases, prolyl oligopeptidase and oligopeptidase B, showed that resonances corresponding to the active center histidine Ndelta1H and Nepsilon2H generally observed in this region, are absent in these enzymes. However, for both enzymes, as well as with the H652A and H652Q active center variants of oligopeptidase B, there are two resonances observed in this region that could be assigned to two protonated histidines with a noncatalytic function. The results indicate that these two histidines participate in strong hydrogen bonds. The absence of resonances pertinent to the active center histidine resonances suggests the absence of a low barrier hydrogen bond between the Asp and His in these two enzymes in their ground states. Addition of the peptide boronic acid t-butoxycarbonyl-(D)Val-Leu-(L)boroArg to oligopeptidase B resulted in potent, slow binding inhibition of the enzyme and the appearance of a new resonance at 15.8 ppm, whose chemical shift is appropriate for a tetrahedral boronate complex and a low barrier hydrogen bond. The results demonstrate important dissimilarities between the active centers of the prolyl oligopeptidase class of serine proteases and the pancreatic and subtilisin classes both in the ground state and in the transition-state analog complexes.

New inhibitors of thrombin and other trypsin-like proteases: hydrogen bonding of an aromatic cyano group with a backbone amide of the P1 binding site replaces binding of a basic side chain.

Highly effective thrombin inhibitors have been obtained by preparing boronic acid analogues of m-cyano-substituted phenylalanine and its incorporation into peptides. The cyano group enhances binding by several orders of magnitude. For example, Ac-(D)Phe-Pro-boroPheOH binds to thrombin with a Ki of 320 nM and the Ki of Ac-(D)Phe-Pro-boroPhe(m-CN)-OH is 0.79 nM. Protein crystal structure determination of trypsin complexed to H-(D)Phe-Pro-boroPhe(m-CN)-OH indicates that the aromatic side chain is bound in the P1 binding site and that the cyano group can act as a H-bond acceptor for the amide proton of Gly219. Enhanced binding for inhibitors containing the m-cyano group was observed for coagulation factor Xa and for the factor VIIa.tissue factor complex [Ki values of Ac-(D)Phe-Pro-boroPhe(mCN)-OH are 760 and 3.3 nM, respectively]. This result is consistent with the sequence homology of these two enzymes in the P1 binding site. Two enzymes lacking the strict homology in the P1 binding site, pancreatic kallikrein and chymotrypsin, did not exhibit significantly enhanced binding.

Activation of the kallikrein-kinin system in arthritis and enterocolitis in genetically susceptible rats: modulation by a selective plasma kallikrein inhibitor.

We have developed models of acute and chronic inflammatory arthritis and enterocolitis using peptidoglycan-polysaccharide injected intraperitoneally or subserosally (intramurally) into the distal ileum and cecum. Acute inflammation occurs in both Buffalo and Lewis rats, characterized by inflammation of the injected areas of the intestine. However, only the genetically susceptible Lewis rat develops chronic synovitis and joint erosion or adhesions and granulomatous enterocolitis. In the Lewis rat but not the Buffalo rat, these changes are accompanied by a decrease in plasma prekallikrein and high-molecular-weight kininogen, reflecting activation of the kallikrein-kinin system. Pretreatment with a specific plasma kallikrein inhibitor modulates the acute and chronic arthritis. The same inhibitor partially abrogates the acute changes characteristic of enterocolitis, and preliminary data suggest similar results in the chronic model. The results of these studies indicate that the kallikrein-kinin system plays an important role in arthritis and enterocolitis induced by bacterial products and that kallikrein inhibitors are potential therapeutic agents for inflammatory arthritis and inflammatory bowel disease.

Cryptosporidium parvum metalloaminopeptidase inhibitors prevent in vitro excystation.

Cryptosporidium parvum arginine aminopeptidase (RAP) was studied during in vitro excystation. Specific RAP inhibitors were identified by using C. parvum extracts. Amastatin, a series of alpha-aminoboronic acids, and the chelating agents EDTA and 1,10-phenanthrolene, but not endoproteinase inhibitors, blocked enzymatic activity. RAP inhibitors found to be effective in soluble enzymatic assays were then studied for their effect on in vitro excystation. 1,10-Phenanthrolene, amastatin, and H-boronorleucine (pinacol) inhibited excystation by 84, 57, and 61%, respectively, compared with solvent-treated control oocysts. Sporozoites remained viable within the oocyst as determined by propidium iodide and fluorescein diacetate dye uptake, suggesting that alpha-aminoboronic acids were not directly lethal to the parasite.

Structure-based understanding of ligand affinity using human thrombin as a model system.

Kinetic study of a series of compounds containing the thrombin-directed peptide D-Phe-ProboroArg-OH had indicated that the structure of the N-terminal blocking group may be correlated with binding [Kettner, C., Mersinger, L., & Knabb, R. (1990) J. Biol. Chem. 265, 18289-18297]. In order to further study this phenomenon, a second series of compounds that contains a C-terminal methyl ester in place of the boronic acid was synthesized, binding measured, and the three-dimensional structure in complex with human thrombin determined by X-ray crystallography. Incubation of Ac-D-Phe-Pro-Arg-OMe, Boc-D-Phe-Pro-Arg-OMe, and H-D-Phe-Pro-Arg-OMe resulted in the formation of thrombin-product complexes within the crystal. Ki values for the corresponding products (free carboxylic acids) were 60 +/- 12 microM, 7.8 +/- 0.1 microM, 0.58 +/- 0.02 microM, respectively, indicating that the nature of the N-terminal blocking group has a significant effect on affinity. Examination of the crystal structures indicated that the higher affinity of the H-D-Phe peptide is due to rearrangement of one residue comprising the S3 site (Glu192) in order to maximize electrostatic interactions with the "NH3(+)-" of H-D-Phe. The relative affinity of Boc-D-Phe-Pro-Arg-OH is due to favorable hydrophobic interactions between thrombin and the bulky butyl group. However, this results in less favorable binding of Arg-P1 in the oxyanion hole as shown by long hydrogen-bonding distances. This work gave rise to some general observations applicable to structure-based drug design: (1) altering the structure of an inhibitor at one site can affect binding at an unchanged distal site; (2) minor alteration of inhibitor structure can lead to small, but significant reorganization of neighboring protein structure; (3) these unexpected reorganizations can define alternate binding motifs.

Selective plasma kallikrein inhibitor attenuates acute intestinal inflammation in Lewis rat.

A specific plasma kallikrein inhibitor, Bz-Pro-Phe-boroArg (P8720), was used to define the relationship between the kallikrein-kinin (K-K) system and acute intestinal inflammation induced by bacterial peptidoglycan-polysaccharide (PG-APS) in Lewis rats. Group I received human serum albumin (HSA) intramurally in the intestine and was treated with HSA. Group II received PG-APS and was treated with P8720. Group III received PG-APS and was treated with HSA. P8720 attenuated the decrease of high-molecular-weight kininogen and factor XI activity (group II vs group III, P < 0.01). P8720 therapy significantly but modestly decreased acute intestinal inflammation measured by gross gut score (P < 0.01) and more dramatically reduced the tissue myeloperoxidase activity (P < 0.05), a measure of granulocyte recruitment, in group II compared with group III. We conclude that the K-K system is directly involved in the pathogenesis of the acute phase of experimental acute inflammation. A specific inhibitor may modulate inflammatory bowel disease.

The S. cerevisiae outwardly-rectifying potassium channel (DUK1) identifies a new family of channels with duplicated pore domains.

Potassium channel subunits have six or two transmembrane segments in addition to a conserved pore-forming (P) domain; four subunits come together to form a channel. A gene was identified in S. cerevisiae (J0911) encoding a protein with eight probable membrane-spanning segments and two such P regions. This protein (Duk1p) is a potassium channel because Xenopus oocytes injected with the corresponding RNA express potassium currents activated by depolarization that are not seen in control oocytes. Similar potassium currents were recorded from wildtype S. cerevisiae spheroplasts, but not from those in which the DUK1 locus had been disrupted. Cells carrying the duk1 delta 1::HIS disruption in addition to a chimeric gene comprising DUK1 behind the GAL1 promoter showed outward currents when grown in galactose, but not when grown in glucose. Additional sequences with the duplicate pore motif were found in C. elegans, suggesting that these proteins represent a novel structural family of potassium channel proteins.