Exploring the potential of boronic acids as inhibitors of OXA‐24/40 β‐lactamase

β‐lactam antibiotics are crucial to the management of bacterial infections in the medical community. Due to overuse and misuse, clinically significant bacteria are now resistant to many commercially available antibiotics. The most widespread resistance mechanism to β‐lactams is the expression of β‐lactamase enzymes. To overcome β‐lactamase mediated resistance, inhibitors were designed to inactivate these enzymes. However, current inhibitors (clavulanic acid, tazobactam, and sulbactam) for β‐lactamases also contain the characteristic β‐lactam ring, making them susceptible to resistance mechanisms employed by bacteria. This presents a critical need for novel, non‐β‐lactam inhibitors that can circumvent these resistance mechanisms. The carbapenem‐hydrolyzing class D β‐lactamases (CHDLs) are of particular concern, given that they efficiently hydrolyze potent carbapenem antibiotics. Unfortunately, these enzymes are not inhibited by clinically available β‐lactamase inhibitors, nor are they effectively inhibited by the newest, non‐β‐lactam inhibitor, avibactam. Boronic acids are known transition state analog inhibitors of class A and C β‐lactamases, and are not extensively characterized as inhibitors of class D β‐lactamases. Importantly, boronic acids provide a novel way to potentially inhibit class D β‐lactamases. Sixteen boronic acids were selected and tested for inhibition of the CHDL OXA‐24/40. Several compounds were identified as effective inhibitors of OXA‐24/40, with K_i values as low as 5 μM. The X‐ray crystal structures of OXA‐24/40 in complex with BA3, BA4, BA8, and BA16 were determined and revealed the importance of interactions with hydrophobic residues Tyr112 and Trp115. These boronic acids serve as progenitors in optimization efforts of a novel series of inhibitors for class D β‐lactamases.     

The effect of a beta‐lactamase inhibitor peptide on bacterial membrane structure and integrity: a comparative study

Co‐administration of beta‐lactam antibiotics and beta‐lactamase inhibitors has been a favored treatment strategy against beta‐lactamase‐mediated bacterial antibiotic resistance, but the emergence of beta‐lactamases resistant to current inhibitors necessitates the discovery of novel non‐beta‐lactam inhibitors. Peptides derived from the Ala46–Tyr51 region of the beta‐lactamase inhibitor protein are considered as potent inhibitors of beta‐lactamase; unfortunately, peptide delivery into the cell limits their potential. The properties of cell‐penetrating peptides could guide the design of beta‐lactamase inhibitory peptides. Here, our goal is to modify the peptide with the sequence RRGHYY that possesses beta‐lactamase inhibitory activity under in vitro conditions. Inspired by the work on the cell‐penetrating peptide pVEC, our approach involved the addition of the N‐terminal hydrophobic residues, LLIIL, from pVEC to the inhibitor peptide to build a chimera. These residues have been reported to be critical in the uptake of pVEC. We tested the potential of RRGHYY and its chimeric derivative as a beta‐lactamase inhibitory peptide on Escherichia coli cells and compared the results with the action of the antimicrobial peptide melittin, the beta‐lactam antibiotic ampicillin, and the beta‐lactamase inhibitor potassium clavulanate to get mechanistic details on their action. Our results show that the addition of LLIIL to the N‐terminus of the beta‐lactamase inhibitory peptide RRGHYY increases its membrane permeabilizing potential. Interestingly, the addition of this short stretch of hydrophobic residues also modified the inhibitory peptide such that it acquired antimicrobial property. We propose that addition of the hydrophobic LLIIL residues to the peptide N‐terminus offers a promising strategy to design novel antimicrobial peptides in the battle against antibiotic resistance. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

Identification of group specific motifs in Beta-lactamase family of proteins

Background  

Beta-lactamases are one of the most serious threats to public health. In order to combat this threat we need to study the molecular and functional diversity of these enzymes and identify signatures specific to these enzymes. These signatures will enable us to develop inhibitors and diagnostic probes specific to lactamases. The existing classification of beta-lactamases was developed nearly 30 years ago when few lactamases were available. DLact database contain more than 2000 beta-lactamase, which can be used to study the molecular diversity and to identify signatures specific to this family.     

alpha-1 Antitrypsin phenotypes determined by isoelectric focusing of the cysteine-antitrypsin mixed disulfide in serum.

The methods available for analysis of sugar kinase activity are usually either complicated or time consuming. Coupled assays, aside from the added cost of coupling enzymes and substrates, present problems due to the pH optima, activators, and inhibitors of the coupling enzymes. Direct separation of the product requires either ion exchange (1) or paper chromatography (2,3). The former requires constant attention and the latter usually takes either overnight for the completion of a chromatogram or a great deal of elution solvent (200 ml) for DEAE paper discs (3).Those enzymes which form phosphorylated products from nonionic substrates (hexokinases, glycerol kinase, phosphoribosyl-transferases, etc.) may be conveniently assayed by chromatograhic separation of a radioactive phosphorylated product from the radioactive nonionic substrate, where the product remains at the origin. In such assays, no interfering coupling enzymes are used and the product can be directly and sensitively measured. The only current limitation with such methods is the time required for the separation of the phosphorylated product. It would be advantageous to obtain the enzyme's activity in as short a time as possible.We present here a method of paper chromatographic separation of phosphorylated product from nonionic substrate which requires only approximately two hours, uses a large petri dish, very little chromatographic grade paper, and almost no attention.     

Molecular mechanisms of cephalosporin resistance in Gram-negative bacteria of the Enterobacteriaceae family]

Extended spectrum beta lactamase genes were detected by the PCR in 87.6% of 231 Enterobacteriaceae strains isolated in medical institutions of Moscow, St. Petersburg, Tomsk and Nazran that showed a decrease in their susceptibility to 3rd generation cephalosporins. Alone or in various combinations TEM type beta lactamases were detected in 43.3% of the isolates, 46.8 and 51.2% of the isolates produced SHV type and CTX type beta lactamases respectively. Combinations of 2 and 3 different determinants were detected in 40 and 14% of the isolates respectively. Production of class C beta lactamases was suspected in 28% of the isolates by their resistance to cefoxitin. The gene of ACT type beta lactamase was detected in 1 strain of Klebsiella pneumoniae and the gene of CMY type beta lactamase was detected in 1 strain of Proteus mirabilis. By the NCCLS 100% of the isolates was susceptible to meropenem, 14% was susceptible to cefotaxime, 64% was susceptible to cefepime, 81% was susceptible to cefoperazone/sulbactam, 47% was susceptible to gentamicin, 57% was susceptible to amikacin and 36% was susceptible to ciprofloxacin.     

Evaluation of deoxygenated oligosaccharide acceptor analogs as specific inhibitors of glycosyltransferases

The glycosyltransferases controlling the biosynthesis of cell-surface complex carbohydrates transfer glycosyl residues from sugar nucleotides to specific hydroxyl groups of acceptor oligosaccharides. These enzymes represent prime targets for the design of glycosylation inhibitors with the potential to specifically alter the structures of cell-surface glycoconjugates. With the aim of producing such inhibitors, synthetic oligosaccharide substrates were prepared for eight different glycosyltransferases. The enzymes investigated were: A, alpha(1----2, porcine submaxillary gland); B, alpha(1----3/4, Lewis); C, alpha(1----4, mung bean); D, alpha(1----3, Lex)-fucosyltransferases; E, beta(1----4)-galactosyltransferase; F, beta(1----6)-N-acetylglucosaminyltransferase V; G, beta(1----6)-mucin-N-acetylglucosaminyltransferase ("core-2" transferase); and H, alpha(2----3)-sialyltransferase from rat liver. These enzymes all transfer sugar residues from their respective sugar nucleotides (GDP-Fuc, UDP-Gal, UDP-GlcNAc, and CMP-sialic acid) with inversion of configuration at their anomeric centers. The Km values for their synthetic oligosaccharide acceptors were in the range of 0.036-1.3 mM. For each of these eight enzymes, acceptor analogs were next prepared where the hydroxyl group undergoing glycosylation was chemically removed and replaced by hydrogen. The resulting deoxygenated acceptor analogs can no longer be substrates for the corresponding glycosyltransferases and, if still bound by the enzymes, should act as competitive inhibitors. In only four of the eight cases examined (enzymes A, C, F, and G) did the deoxygenated acceptor analogs inhibit their target enzymes, and their Ki values (all competitive) remained in the general range of the corresponding acceptor Km values. No inhibition was observed for the remaining four enzymes even at high concentrations of deoxygenated acceptor analog. For these latter enzymes it is suggested that the reactive acceptor hydroxyl groups are involved in a critical hydrogen bond donor interaction with a basic group on the enzyme which removes the developing proton during the glycosyl transfer reaction. Such groups are proposed to represent logical targets for irreversible covalent inactivation of this class of enzyme.     

Characterization of two carnosine-degrading enzymes from rat brain. Partial purification and characterization of a carnosinase and a beta-alanyl-arginine hydrolase

From rat brain extracts, two carnosine-degrading enzymes have been identified and partially purified by ion-exchange chromatography, hydrophobic interaction chromatography on phenyl-Sepharose CL-4B and gel filtration. These enzymes exhibit distinct differences in their chemical characteristics and substrate specificities. One enzyme, designated carnosinase, preferentially hydrolyzes carnosine and exhibits a low Km value (0.02 mM) towards this substrate. Carnosinase also degrades anserine but not homocarnosine or homoanserine. The other carnosine-degrading enzyme hydrolyzes beta Ala-Arg considerably faster than carnosine and, therefore, has been tentatively designated beta Ala-Arg hydrolase. This enzyme exhibits high Km values with carnosine (Km = 25 mM) and beta Ala-Arg (Km = 2 mM). Homocarnosine and gamma-aminobutyryl-arginine are not degraded by beta Ala-Arg hydrolase. Neither enzyme is inhibited by agents reactive on activated hydroxyl groups, such as diisopropyl fluorophosphate, and also not by a variety of peptidase inhibitors of microbial origin or from other sources. Carnosinase is also not inhibited by bestatin but beta Ala-Arg hydrolase, although not an aminopeptidase, is strongly inhibited by this aminopeptidase inhibitor (IC50 = 50 nM). While carnosinase is strongly inhibited by thiol-reducing agents such as dithioerythritol and 2-mercaptoethanol, beta Ala-Arg hydrolase is stabilized and activated by these substances. Both enzymes are strongly inhibited by metal-chelating agents. Carnosinase, however, is not dependent on exogeneously added metal ions and is strongly inhibited by Mn2+ as well as by heavy metal ions. In contrast, beta Ala-Arg hydrolase requires Mn2+ ions for full enzymatic activity. Based on these differences, selective incubation conditions could be evaluated in order to determine specifically both enzyme activities in crude tissue extracts. In rat, both enzymes are present in all tissues tested, except skeletal muscles, but considerable differences in their relative distribution among different tissues are also observed.     

Analytical isoelectric focusing of R factor-determined beta-lactamases: correlation with plasmid compatibility.

R factor-determined beta-lactamases have been investigated by analytical isoelectric focusing. The enzymes such as those specified by the R6K and RP4 plasmids (TEM-type enzymes) are notably homogenous in biochemical tests (Hedges et al., 1974), but two subclasses can be distinguished by isoelectric focusing. Three subclasses can be distinguished among the oxacillin-hydrolyzing enzymes, in good agreement with the classification based upon biochemical characteristics (Dale and Smith, 1974). The TEM-type beta-lactamases are promiscuously distributed among plasmids of a wide variety of compatibility groups, whereas the various oxacillin-hydrolyzing enzymes show some degree of correlation with compatibility.     

Selectivity of inhibitors of endocannabinoid biosynthesis evaluated by activity-based protein profiling

The endocannabinoid 2-arachidonoylglycerol (2-AG) has been implicated as a key retrograde mediator in the nervous system based on pharmacological studies using inhibitors of the 2-AG biosynthetic enzymes diacyglycerol lipase alpha and beta (DAGL-alpha/beta). Here, we show by competitive activity-based protein profiling that the DAGL-alpha/beta inhibitors, tetrahydrolipstatin (THL) and RHC80267, block several brain serine hydrolases with potencies equal to or greater than their inhibitory activity against DAGL enzymes. Interestingly, a minimal overlap in target profiles was observed for THL and RHC80267, suggesting that pharmacological effects observed with both agents may be viewed as good initial evidence for DAGL-dependent events.     

Theaflavin‐3,3´‐digallate increases the antibacterial activity of β‐lactam antibiotics by inhibiting metallo‐β‐lactamase activity

Metallo‐β‐lactamases (MBLs) are some of the best known β‐lactamases produced by common Gram‐positive and Gram‐negative pathogens and are crucial factors in the rise of bacterial resistance against β‐lactam antibiotics. Although many types of β‐lactamase inhibitors have been successfully developed and used in clinical settings, no MBL inhibitors have been identified to date. Nitrocefin, checkerboard and time‐kill assays were used to examine the enzyme behaviour in vitro. Molecular docking calculation, molecular dynamics simulation, calculation of the binding free energy and ligand‐residue interaction decomposition were used for mechanistic research. The behaviour of the enzymes in vivo was investigated by a mouse infection experiment. We showed that theaflavin‐3,3´‐digallate (TFDG), a natural compound lacking antibacterial activities, can inhibit the hydrolysis of MBLs. In the checkerboard and time‐kill assays, we observed a synergistic effect of TFDG with β‐lactam antibiotics against methicillin‐resistant Staphylococcus aureus BAA1717. Molecular dynamics simulations were used to identify the mechanism of the inhibition of MBLs by TFDG, and we observed that the hydrolysis activity of the MBLs was restricted by the binding of TFDG to Gln242 and Ser369. Furthermore, the combination of TFDG with β‐lactam antibiotics showed effective protection in a mouse Staphylococcus aureus pneumonia model. These findings suggest that TFDG can effectively inhibit the hydrolysis activity of MBLs and enhance the antibacterial activity of β‐lactam antibiotics against pathogens in vitro and in vivo.     

Analysis of the plasticity of location of the Arg244 positive charge within the active site of the TEM‐1 β‐lactamase

A large number of β‐lactamases have emerged that are capable of conferring bacterial resistance to β‐lactam antibiotics. Comparison of the structural and functional features of this family has refined understanding of the catalytic properties of these enzymes. An arginine residue present at position 244 in TEM‐1 β‐lactamase interacts with the carboxyl group common to penicillin and cephalosporin antibiotics and thereby stabilizes both the substrate and transition state complexes. A comparison of class A β‐lactamase sequences reveals that arginine at position 244 is not conserved, although a positive charge at this structural location is conserved and is provided by an arginine at positions 220 or 276 for those enzymes lacking arginine at position 244. The plasticity of the location of positive charge in the β‐lactamase active site was experimentally investigated by relocating the arginine at position 244 in TEM‐1 β‐lactamase to positions 220, 272, and 276 by site‐directed mutagenesis. Kinetic analysis of the engineered β‐lactamases revealed that removal of arginine 244 by alanine mutation reduced catalytic efficiency against all substrates tested and restoration of an arginine at positions 272 or 276 partially suppresses the catalytic defect of the Arg244Ala substitution. These results suggest an evolutionary mechanism for the observed divergence of the position of positive charge in the active site of class A β‐lactamases.     

Thiomandelic acid, a broad spectrum inhibitor of zinc beta-lactamases: kinetic and spectroscopic studies

Resistance to beta-lactam antibiotics mediated by metallo-beta-lactamases is an increasingly worrying clinical problem. Candidate inhibitors include mercaptocarboxylic acids, and we report studies of a simple such compound, thiomandelic acid. A series of 35 analogues were synthesized and examined as metallo-beta-lactamase inhibitors. The K(i) values (Bacillus cereus enzyme) are 0.09 microm for R-thiomandelic acid and 1.28 microm for the S-isomer. Structure-activity relationships show that the thiol is essential for activity and the carboxylate increases potency; the affinity is greatest when these groups are close together. Thioesters of thiomandelic acid are substrates for the enzyme, liberating thiomandelic acid, suggesting a starting point for the design of "pro-drugs." Importantly, thiomandelic acid is a broad spectrum inhibitor of metallo-beta-lactamases, with a submicromolar K(i) value for all nine enzymes tested, except the Aeromonas hydrophila enzyme; such a wide spectrum of activity is unprecedented. The binding of thiomandelic acid to the B. cereus enzyme was studied by NMR; the results are consistent with the idea that the inhibitor thiol binds to both zinc ions, while its carboxylate binds to Arg(91). Amide chemical shift perturbations for residues 30-40 (the beta(3)-beta(4) loop) suggest that this small inhibitor induces a movement of this loop of the kind seen for other larger inhibitors.     

Growth regulatory peptide production by human breast carcinoma cells

The mechanisms by which human breast cancers regulate their own growth have been studied by us in an in vitro model system. We showed that specific growth factors (IGF-I, TGF alpha, PDGF) are secreted by human breast cancer cells. A variety of experiments suggest that they are involved in tumor growth and progression. These activities are induced by estradiol in hormone-dependent breast cancer cells and secreted constitutively by estrogen-independent cells. Concentrates of conditioned medium derived from breast cancer cells can induce the growth of hormone-dependent cells in vivo in athymic nude mice. Hormone-dependent breast cancer cells also secrete TGF beta. TGF beta is growth inhibitory. Growth inhibitors such as antiestrogens or glucocorticoids increase TGF beta secretion. An antiestrogen-resistant mutant of MCF-7 cells does not secrete TGF beta when treated with antiestrogen, but is growth inhibited when treated with exogenous TGF beta. Thus, TGF beta functions as a negative autocrine growth regulator and is probably responsible for some of the growth inhibitory effects of antiestrogens.     

Hydrogen bonding in steroidogenesis: studies on new heterocyclic analogs of estrone that inhibit human estradiol 17 beta-dehydrogenase

New heterocyclic analogs of estrone are reported that inhibit estradiol 17 beta-dehydrogenase (E2-17 beta DH) from human placenta. The inhibitors are efficiently synthesized in two steps from estrone (or its 3-O-methyl ether), giving fully characterized analogs with pyrazole or isoxazole fused to the 16,17-position on the D ring. Dixon plots of enzyme kinetic data show the heterocyclic steroids are competitive inhibitors of E2-17 beta DH. Correlating molecular structures of the inhibitors with their Ki-values yields a pattern suggesting intermolecular hydrogen bonding stabilizes the [(pyrazole)inhibitor-E2-17 beta DH] complexes. A free energy difference of 2.74 Kcal/mol calculated from Ki-value differences between hydrogen bonded (4.08 microM) and non-bonded (425 microM) [inhibitor-E2-17 beta DH] complexes is in the range for intermolecular hydrogen bonding. We conclude that specific intermolecular hydrogen bonds stabilize [hydroxysteroid-enzyme] complexes, thereby making important contributions to the affinity between hydroxysteroids and steroid-specific enzymes of steroidogenesis.     

Isoform-specific phosphoinositide 3-kinase inhibitors from an arylmorpholine scaffold

Phosphoinositide 3-kinases (PI3-Ks) are an ubiquitous class of signaling enzymes that regulate diverse cellular processes including growth, differentiation, and motility. Physiological roles of PI3-Ks have traditionally been assigned using two pharmacological inhibitors, LY294002 and wortmannin. Although these compounds are broadly specific for the PI3-K family, they show little selectivity among family members, and the development of isoform-specific inhibitors of these enzymes has been long anticipated. Herein, we prepare compounds from two classes of arylmorpholine PI3-K inhibitors and characterize their specificity against a comprehensive panel of targets within the PI3-K family. We identify multiplex inhibitors that potently inhibit distinct subsets of PI3-K isoforms, including the first selective inhibitor of p110beta/p110delta (IC(50) p110beta=0.13 microM, p110delta=0.63 microM). We also identify trends that suggest certain PI3-K isoforms may be more sensitive to potent inhibition by arylmorpholines, thereby guiding future drug design based on this pharmacophore.     

Potential role of glycosidase inhibitors in industrial biotechnological applications

The nutrient content of food and animal feed may be improved through new knowledge about enzymatic changes in complex carbohydrates. Enzymatic hydrolysis of complex carbohydrates containing alpha or beta glycosidic bonds is very important in nutrition and in several technological processes. These enzymes are called glycosidases (Enzyme Class 3.2.1) and include amylases, pectinases and xylanases. They are present in many foods such as cereals, but their microbial analogues are often produced and added in many food processes, for instance to improve the shelf-life of bakery products, clear beer, produce glucose, fructose or dextrins, hydrolyse lactose, modify food pectins, or improve processes. However, many plant foods also contain endogenous inhibitors, which reduce the activity of glycosidases, in particular, proteins, peptides, complexing agents and phenolic compounds. The plant proteinaceous inhibitors of glycosidases are in focus in this review whose objective is to report the effect and implications of these inhibitors in industrial processes and applications. These studies will contribute to the optimisation of industrial processes by using modified enzymes not influenced by the natural inhibitors. They will also allow careful selection of raw material and reaction conditions, and future development of new genetic varieties low in inhibitors. These are all new and very promising concepts for the food and feed sector.     

'Beta-lactams' as beta-lactamase inhibitors

The application of inhibitors to block the beta-lactamase destruction of penicillins and cephalosporins by resistant bacteria is a potentially useful way of improving the efficacy of established compounds. Certain semi-synthetic penicillins and cephalosporins have been found to be competitive inhibitors of selected beta-lactamases but an examination of streptomycete culture fluids has revealed two new types of beta-lactam compound: clavulanic acid, which is a progressive inactivator of a wide range of beta-lactamases, and the olivanic acids, which are both broad-spectrum antibiotics and potent beta-lactamase inhibitors. Penicillanic acid sulphone and 6-beta-bromopenicillanic acid have been shown to be significant inhibitors of beta-lactamase. The chemotherapeutic application of these compounds is discussed.     

The role of the non-conserved residue at position 104 of class A beta-lactamases in susceptibility to mechanism-based inhibitors

The role of the non-conserved amino acid residue at position 104 of the class A beta-lactamases, which comprises a highly conserved sequence of amino acids at the active sites of these enzymes, in both the hydrolysis of beta-lactam substrates and inactivation by mechanism-based inhibitors was investigated. Site-directed mutagenesis was performed on the penPC gene encoding the Bacillus cereus 569/H beta-lactamase I to replace Asp104 with the corresponding Staphylococcus aureus PC1 residue Ala104. Kinetic data obtained with the purified Asp104Ala B. cereus 569/H beta-lactamase I was compared to that obtained from the wild-type B. cereus and S. aureus enzymes. Replacement of amino acid residue 104 had little effect on the Michaelis parameters for the hydrolysis of both S- and A-type penicillins. Relative to wild-type enzyme, the Asp104Ala beta-lactamase I had 2-fold higher Km values for benzylpenicillin and methicillin, but negligible difference in Km for ampicillin and oxacillin. However, kcat values were also slightly increased resulting in little change in catalytic efficiency, kcat/Km. In contrast, the Asp104Ala beta-lactamase I became more like the S. aureus enzyme in its response to the mechanism-based inhibitors clavulanic acid and 6-beta-(trifluoromethane sulfonyl)amido-penicillanic acid sulfone with respect to both response to the inhibitors and subsequent enzymatic properties. Based on the known three-dimensional structures of the Bacillus licheniformis 749/C, Escherichia coli TEM and S. aureus PC1 beta-lactamases, a model for the role of the non-conserved residue at position 104 in the process of inactivation by mechanism-based inhibitors is proposed.     

Equivalence of branched and unbranched Michaelian pathways concerning periodic signal transmission

Sinusoidal oscillation transmission through branched metabolic pathways is studied. Two systems are analyzed, which are composed of two convergent reaction branches and differ in the length of one of them. Linear kinetics is assumed first. Michaelis-Menten enzymes are then considered by using previous results that suggest their behavior with respect to propagation of oscillations is close to linearity around the mean input flux. As a result, there exist ways to modulate the activity of the enzymes so that propagation is equivalent for branched and specific unbranched pathways. Cells may have taken advantage of such a possibility in cases where oscillations have a biological role.     

Purification and characterization of two alpha-amylase inhibitors from seeds of tepary bean (Phaseolus acutifolius A. Gray)

Two proteinaceous alpha-amylase inhibitors termed alphaAI-Pa1 and alphaAI-Pa2 were purified from seeds of a cultivated tepary bean (Phaseolus acutifolius A. Gray, cv. PI311897). The two inhibitors differed in their specificity towards alpha-amylases of insect pests such as bruchids, although neither showed any inhibitory activity against alpha-amylases of mammalian, bacterial or fungal origin. AlphaAI-Pa2 resembles two common bean inhibitors, alphaAI-1 and alphaAI-2, in several characteristics such as N-terminal amino acid sequences and oligomeric structure being composed of alpha and beta subunits. In contrast alphaAI-Pa1 is composed of a single glycopolypeptide with a molecular mass of 35 kDa, and its N-terminal amino acid sequence resembled that of seed lectins in tepary bean and common bean. The information on the two tepary bean alpha-amylase inhibitors may be useful not only for providing insight into critical structure for the specificity towards different alpha-amylase enzymes but also for enhancing insect resistance in crops.