Crystallographic structure of a complex between trypsin and a nonapeptide derived from a Bowman-Birk inhibitor found in Vigna unguiculata seeds.

Natural inhibitors of proteases have been classified into different families, among them is the Bowman-Birk Inhibitor (BBI) family. Members of BBI have two structurally reactive loops that simultaneously inhibit trypsin and chymotrypsin. Here, we have investigated the binding of bovine trypsin by a cyclic nonapeptide, named PTRY9 (CTKSIPPQC), derived of the black-eyed pea trypsin/chymotrypsin inhibitor (BTCI) from Vigna unguiculata seeds. This peptide was synthetically produced with the disulfide bond restraining its conformation to mimic the reactive loop that inhibits trypsin. PTRY9 complexed to pancreatic bovine trypsin was crystallized in orthorhombic and trigonal space groups, P212121 and P3221, with maximum resolutions of 1.15 and 1.61 Å, respectively. The structures presented refinement parameters of Rwork = 14.52 % and Rfree = 15.59 %; Rwork = 15.60 % and Rfree = 18.78 %, and different surface area between the peptide and the enzyme of 1024 Å2 and 1070 Å2, respectively. The binding site of the PTRY9 is similar to that found for BTCI as shown by a r.m.s.d. of 0.358 Šbetween the superimposed structures and the electrostatic complementary pattern at the enzyme-peptide interface. Additionally, enzyme inhibition assays show that the affinity of trypsin for PTRY9 is smaller than that for BTCI. In vitro assays revealed that, like BTCI, this synthetic peptide is not cytotoxic for normal mammary epithelial MCF-10A cells, but exerts cytotoxic effects on MDA.MB.231 invasive human breast cancer cells.

Development of a time-resolved fluorescence resonance energy transfer assay for cyclin-dependent kinase 4 and identification of its ATP-noncompetitive inhibitors.

Protein kinases are recognized as important drug targets due to the pivotal roles they play in human disease. Many kinase inhibitors are ATP competitive, leading to potential problems with poor selectivity and significant loss of potency in vivo due to cellular ATP concentrations being much higher than K(m). Consequently, there has been growing interest in the development of ATP-noncompetitive inhibitors to overcome these problems. There are challenges to identifying ATP-noncompetitive inhibitors from compound library screens because ATP-noncompetitive inhibitors are often weaker and commonly excluded by potency-based hit selection criteria in favor of abundant and highly potent ATP-competitive inhibitors in screening libraries. Here we report the development of a time-resolved fluorescence resonance energy transfer (TR-FRET) assay for protein kinase cyclin-dependent kinase 4 (CDK4) and the identification of ATP-noncompetitive inhibitors by high-throughput screening after employing a strategy to favor this type of inhibitors. We also present kinetic characterization that is consistent with the proposed mode of inhibition.

Advantageous use of SFC for separation of crude therapeutic peptides and peptide libraries.

A simple SFC/MS (Supercritical Fluid Chromatography/Mass Spectrometry) method set was developed to effectively screen separations of various crude synthetic peptide products of pharmaceutical interest. Additives to the modifier methanol which were successful for these separations were found to include TFA (trifluoroacetic acid) and ammonia mixed with TFA, each at 0.1 % (v/v) composition in methanol. A final screening column set consisted of 2-ethylpyride (2-EP), 4-ethylpyridine (4-EP) and cross-linked diol (Luna™ HILIC) stationary phases. Small, linear and macrocyclic peptides with fewer than ten residues could all be eluted with good performance under at least one of the method conditions comprising the final screening protocol. For larger peptides either the 4-EP and HILIC columns with the above additives provided a good initial screening method set without 2-EP. The gradient was slightly longer and shifted to higher polarity relative to the gradients for smaller peptides. This method was often successful to elute large, hydrophilic peptides up to a 41-mer with acceptable peak profiles although the largest peptides with most ionizable residues were most challenging. For these peptides generally HILIC column performance was better with TFA + ammonia in the modifier than with TFA, while 4-EP performance was usually improved with TFA relative to TFA + ammonia.

[Hip fractures in the elderly. Observational study in an unit of intermediate care]. / Frattura del collo del femore negli anziani. Studio osservazionale in una Lungodegenza riabilitativa.

ASL TO4-ICU inpatients with hip fracture over 5 month-period were 45, 12 men and 33 women, average age 81. In 42 patients the fall was accidental or environment-related, and in 40 cases it occurred at home. More than 3 coexisting diseases were found in 22 patients (48%), and polipharmacotherapy with more than 3 drugs in 27 (60%). Only 4 (8%) patients presented a diagnosis of osteoporosis, and only one treated with antiosteoporotic drugs. Before the fracture occurred, 35 (77%) subjects walked without help; 28 (62%) were functionally independent, 17 (38%) dependent; cognitive impairment was diagnosed in 11 (24%) patients. Side-fracture was intracapsular in 17 (38%), extracapsular in 28 (62%). Surgery treatment was osteosinthesys in 26 (58%), endoprosthesis in 11 (24%), total hip prosthesis in 8 (18%). Surgery-timing was of 3 or more days in 23 (51%) patients. In the elderly osteoporosis is underdiagnosed and undertreated, and surgery of hip fracture is always delayed.

Crystal structure of the Bowman-Birk Inhibitor from Vigna unguiculata seeds in complex with beta-trypsin at 1.55 A resolution and its structural properties in association with proteinases.

The structure of the Bowman-Birk inhibitor from Vigna unguiculata seeds (BTCI) in complex with beta-trypsin was solved and refined at 1.55 A to a crystallographic R(factor) of 0.154 and R(free) of 0.169, and represents the highest resolution for a Bowman-Birk inhibitor structure to date. The BTCI-trypsin interface is stabilized by hydrophobic contacts and hydrogen bonds, involving two waters and a polyethylene glycol molecule. The conformational rigidity of the reactive loop is characteristic of the specificity against trypsin, while hydrophobicity and conformational mobility of the antichymotryptic subdomain confer the self-association tendency, indicated by atomic force microscopy, of BTCI in complex and free form. When BTCI is in binary complexes, no significant differences in inhibition constants for producing a ternary complex with trypsin and chymotrypsin were detected. These results indicate that binary complexes present no conformational change in their reactive site for both enzymes confirming that these sites are structurally independent. The free chymotrypsin observed in the atomic force microscopy assays, when the ternary complex is obtained from BTCI-trypsin binary complex and chymotrypsin, could be related more to the self-association tendency between chymotrypsin molecules and the flexibility of the reactive site for this enzyme than to binding-related conformational changes.

Crystallization, data collection and processing of the chymotrypsin-BTCI-trypsin ternary complex.

A ternary complex of the black-eyed pea trypsin and chymotrypsin inhibitor (BTCI) with trypsin and chymotrypsin was crystallized by the sitting-drop vapour-diffusion method with 0.1 M HEPES pH 7.5, 10%(w/v) polyethylene glycol 6000 and 5%(v/v) 2-methyl-2,4-pentanediol as precipitant. BTCI is a small protein with 83 amino-acid residues isolated from Vigna unguiculata seeds and is able to inhibit trypsin and chymotrypsin simultaneously by forming a stable ternary complex. X-ray data were collected from a single crystal of the trypsin-BTCI-chymotrypsin ternary complex to 2.7 A resolution under cryogenic conditions. The structure of the ternary complex was solved by molecular replacement using the crystal structures of the BTCI-trypsin binary complex (PDB code 2g81) and chymotrypsin (PDB code 4cha) as search models.

Thermodynamic basis for antibody binding to Z-DNA: comparison of a monoclonal antibody and its recombinant derivatives.

Antibody engineering represents a promising area in biotechnology. Recombinant antibodies can be easily manipulated generating new ligand and effector activities that can be used as prototype magic bullets. On the other hand, an extensive knowledge of recombinant antibody binding and stability features are essential for an efficient substitution. In this study, we compared the stability and protein binding properties of two recombinant antibody fragments with their parental monoclonal antibody. The recombinant fragments were a monomeric scFv and a dimeric one, harboring human IgG1 CH2-CH3 domains. We have used fluorescence titration quenching to determine the thermodynamics of the interaction between an anti-Z-DNA monoclonal antibody and its recombinant antibody fragments with Z-DNA. All the antibody fragments seemed to bind DNA similarly, in peculiar two-affinity states. Enthalpy-entropy compensation was observed for both affinity states, but a marked entropy difference was observed for the monomeric scFv antibody fragment, mainly for the high affinity binding. In addition, we compared the stability of the dimeric antibody fragment and found differences favoring the monoclonal antibody. These differences seem to derive from the heterologous expression system used.

Oligomerization states of Bowman-Birk inhibitor by atomic force microscopy and computational approaches.

Several methods have been applied to study protein-protein interaction from structural and thermodynamic point of view. The present study reveals that atomic force microscopy (AFM), molecular modeling, and docking approaches represent alternative methods offering new strategy to investigate structural aspects in oligomerization process of proteinase inhibitors. The topography of the black-eyed pea trypsin/chymotrypsin inhibitor (BTCI) was recorded by AFM and compared with computational rigid-bodies docking approaches. Multimeric states of BTCI identified from AFM analysis showed globular-ellipsoidal shapes. Monomers, dimers, trimers, and hexamers were the most prominent molecular arrays observed in AFM images as evaluated by molecular volume calculations and corroborated by in silico docking and theoretical approaches. We therefore propose that BTCI adopts stable and well-packed self-assembled states in monomer-dimer-trimer-hexamer equilibrium. Although there are no correlation between specificity and packing efficiency among proteinases and proteinase inhibitors, the AFM and docked BTCI analyses suggest that these assemblies may exist in situ to play their potential function in oligomerization process.

Relationship between Prostatic Specific Antigen (PSA) and volume of the prostate in the Benign Prostatic Hyperplasia in the elderly.

Increase of the Prostatic Specific Antigen (PSA) is a non-invasive, sensitive and specific markers for prostatic diseases, including prostatic cancer. However, age-related Benign Prostatic Hyperplasia (BPH), as well as prostatitis, may at the same time alter PSA values. The aim of this study was to evaluate the relationship between ageing and PSA, and whether age-specific upper normal limits of PSA should be considered for elderly patients. We evaluated 569 consecutive subjects aged 60 years or more (mean age 74.2 years) who were free from malignant prostatic disease, without clinical evidence of prostatic phlogosis and who were not receiving PSA levels affecting drugs. All patients underwent Digital Rectal Examination (DRE) and Trans-Rectal Ultrasonography (TRU), with determination of the three prostatic diameters, the Maximum Adenoma Diameter (MAD) and calculation of the prostatic volume (PV) by the ellipsoid formula. PSA was determined in all patients before DRE and TRU, and the PSA free ratio was determined in those with total PSA values >4 ng/ml. The PSA density was calculated according to the formula PSA/PV. One hundred and seventy-nine subjects (31.6%) were found to have PSA values >4 ng/ml: among them, 26 (14.5%) had values exceeding 10 ng/ml. Age was slightly correlated with PV (P<0.05), but not with PSA values. On the contrary, PSA values were strongly related with PV and MAD (P<0.01 both). Mean PSA-free ratio was 16.3+/-6.0% and most of patients had values in the so-called 'grey zone' of discrimination between benignity and malignity. Elevated PSA levels are common in older subjects without evidence of prostatic malignancy; PSA values are poorly affected by age itself and strongly correlated with increasing PV. These results suggest the possibility to consider as indicative of benignity PSA values between 4 and 10 ng/ml, when these values are associated with relevant increase of PV and with PSA-free ratio greater than 10%.

High-throughput preparative process utilizing three complementary chromatographic purification technologies.

A high-throughput process was developed in which wells in plates generated from parallel synthesis are automatically channeled to an appropriate purification technique using analytical data as a guide. Samples are directed to either of three fundamentally different preparative techniques: HPLC with UV-triggered fraction collection, supercritical fluid chromatography (SFC) with UV-triggered fraction collection, or HPLC with MS-triggered fraction collection. Automated analysis of the analytical data identifies the product compound mass and creates work lists based on chromatographic properties exhibited in the data so that each preparative instrument cherry picks the appropriate list of samples to purify when a preparative-scale plate is loaded.

Ammonia as a preferred additive in chiral and achiral applications of supercritical fluid chromatography for small, drug-like molecules.

Supercritical fluid chromatography is routinely utilized by analytical separations groups in the pharmaceutical industry to efficiently handle separations for discovery medicinal chemistry purposes. Purifications are performed on samples ranging from a few milligrams up to hundreds of grams. Basic additives dissolved into the liquid component of the SFC mobile phase are commonly used to improve peak shape and efficiency in achiral and chiral separations. While for purposes of analysis there is minimal consequence to additive introduction in the mobile phase, for preparative separations one needs to consider the potential effect of an additive's presence when concentrated with the desired compound. Following an SFC purification using an additive-containing modifier, the resulting fractions will contain an easily evaporated modifier, and after its evaporation perhaps still significant levels of the less volatile additive. Depending on the aqueous solubility and basicity of the final product, the process of removing basic amine additives can be time-consuming and can result in reduced yields. NMR analysis following preparative isolation and evaporation often reveals the fact of insufficient removal of the chromatographic additive even after aqueous work up steps. In this study, ammonia is evaluated as an alternative additive to strong bases such as diethylamine (DEA) in SFC purification and analysis and to the authors' knowledge no previous publication has been written describing the application of methanolic ammonia as an additive for SFC separations. Dimethylethylamine (DMEA), a more volatile additive than DEA, is also evaluated relative to ammonia for its potential to simplify the isolation process after purification and in terms of chromatographic performance. The loss in concentration of ammonia in methanol modifier over time due to evaporation and effects of that loss are also described. Furthermore, for ammonia the analytical benefit is shown to extend to on-line mass spectrometric detection relative to other basic additives.

Preserving the chromatographic integrity of high-speed supercritical fluid chromatography separations using time-of-flight mass spectrometry.

The advantage of high-speed time-of-flight-mass spectrometry (TOF-MS) detection for ultrafast qualitative supercritical fluid chromatography/mass spectrometry (SFC/MS) applications allows the superior resolving power of SFC to be exploited in high-throughput analysis. A chromatographic comparison of quadrupole MS and TOF-MS shows high-speed TOF total ion current data point sampling to be more indicative of fast SFC separations and corresponding short (1-2 s) baseline peak widths. Results shown for analysis of a six-compound mixture with two peaks eluting at 0.86 and 0.89 min exhibit >50% resolution by high-speed TOF data sampling, whereas the same peaks appear to coelute using quadrupole MS data sampling. Additionally, a marked improvement in the peak baseline widths is afforded by fast TOF data acquisition of 0.1 s/spectrum, resulting in a reduction in the baseline width, 1.6 s, of sulfanilamide in a four-compound mixture that is more than 2-fold greater than that achieved at the slower data acquisition of 0.5 s/spectrum. The resulting increase in resolution and improved peak shapes allow automatic integration routines to perform more effectively. For most classes of compounds amenable to high performance liquid chromatography, including druglike species, steroids, and polymers, the union of SFC with TOF-MS provides the maximum density of chemical information per unit time available with any high-speed chromatographic/mass spectrometric method.

A mass spectrometry plate reader: monitoring enzyme activity and inhibition with a Desorption/Ionization on Silicon (DIOS) platform.

A surface-based laser desorption/ionization mass spectrometry assay that makes use of Desorption/Ionization on Silicon Mass Spectrometry (DIOS-MS) has been developed to monitor enzyme activity and enzyme inhibition. DIOS-MS has been used to characterize inhibitors from a library and then to monitor their activity against selected enzyme targets, including proteases, glycotransferase, and acetylcholinesterase. An automated DIOS-MS system was also used as a high-throughput screen for the activity of novel enzymes and enzyme inhibitors. On two different commercially available instruments, a sampling rate of up to 38 inhibitors per minute was accomplished, with thousands of inhibitors being monitored. The ease of applying mass spectrometry toward developing enzyme assays and the speed of surface-based assays such as DIOS for monitoring inhibitor effectiveness and enzyme activity makes it attractive for a broad range of screening applications.

Structural similarity in Bowman-Birk type protease inhibitors based on hydrophobicity

The degree of similarity in the three-dimensional structures of thirteen legume Bowman-Birk type protease inhibitors has been examined on the basis of the patterns of hydrophobicity found in their amino acid sequences, following the procedure described by Sweet & Eisenberg (1983). In the group of such double-headed protease inhibitors two sub-groups are distinguished presenting high structural similarity among their respective members and low similarity between them. Phylogenetic trees have been constructed from hydrophobicity difference and minimum mutational distance matrices, respectively

Phospholipases A2: a dendrogram from a distance matrix based on size and hydrophobicity of the residues in their homologous sequences

A distance matrix was obtained from aligned homologous sequences of 32 phospholipases A2 (EC 3.1.1.4) (24 from Elapid and 5 from Viperid venoms, and 3 from amammals), on the basis of the quantities Dij which are defined from a two-dimensional vector representation of the amino acid residues (dimensions: size and hydrophobicity). These Dij quantities were proposed in a previous paper (Ventura, M.M., (1989), An. Acad. brasil. Ci., 61:215). A dendrogram was constructed from this distance matrix empoying, for cluster analysis, the unweighted pair-group using arithmetic averages. Two groups of phospholipases A2: a) Elapid venom enzymes together with the three mammalian pancreatic enzymes (bovine, equine and porcine), and b) Viper venom enzymes (Crotalus, Trimeresurus and Bitis enzymes) can be well distinguished in the topology of the dendrogram. The Elapid group of enzymes in divided into two subgroups: a) Naja, Hemachatus and Bungarus venom enzymes, and b) Notechis, Laticauda, Enhydrina pancreatic phospholipases A2 and the enzymes from Naja, Hemachatus and Bungarus. These results are similar to those reported by Dufton and Hider (Eur. J. Biochem., 137:545 (1983)) which were obtained from the distance matrix based on minimum mutation distance between 25 selected residue positions in the pairwise compared sequences

Electrostatic interactions of charged ionizable groups with the single histidine residue in lysozyme

Foram calculadas as interaçöes eletrostáticas de grupos ionizáveis carregados positiva e negativamente com o resíduo único de histidina, em lisozima de clara de ovo, por meio de um modelo dielétrico uniforme. Na medida em que os átomos envolvidos säo expostos a um solvente ou se localizam próximo à superfície da proteína, foram consideradas somente as interaçöes mediadas pelo solvente (E=80). Foi calculada também a energia total de interaçäo G=1,84 Kcal.mol-1, equivalendo a pK=-1,34. Admitindo-se um pK=7,0 näo pertubado (intrínseco) para o resíduo de histidina, obtém-se um pK aparente de 5,7, valor de acordo com o de 5,8 encontrado na literatura. Esse procedimento simplificado é aplicável a outras proteínas que possuem grupos ionizáveis carregados expostos a solventes, com o intuito de calcular as interaçöes eletrostáticas e determinar sua influência na ionizaçäo de resíduos de aminoácidos nessas proteínas

Do snake venom cytoloxins eschibit amphiphilicity?

It is suggested, on the basis of the structural information available from the literature, that the molecules of cobramine B and homologous cytotoxins, in contrast to snake venom neurotoxins, are amphiphilic in the sense that they are composed of a predominantly hydrophobic multi-stranded ß-sheet and other regions sharply hydrophilic. It is possible that the direct lytic activity of snake venom cytotoxins is due, at least in part, to their amphiphathy

Phylogenetic relationships of snake venom toxic proteins

Two kinds of distance matrices have been formed from minimum mutational distances and absolute hydrophobicity differences obtained by comparison of aligned homologous sequences of 56 toxins from venom os snakes belonging to 7 genera. Phylogenetic trees were constructed from these distance matrices, employing the unweighted pair-group method using arthmetic averages (UPGMA). The Pearson product-moment correlation coefficient has been used to estimate the agreement between the original distance matrix and that obtained directly from the dendrogram. For al these procedures the set of computer programs PHYTREE (written in BASIC for micro-computer, and available from the author) has been used

Secondary structure of soybean trypsin inhibitor (Kunitz): a study by infrared spectroscopy

The infrared spectrum (amide I'region) of Kunitz soybean trypsin inhibitor (SBTI) was obtained in D2O solution and resolved into Gaussian componentes. A prominent broad band centered at 1643 cm -1 is shown on the unresolved spectrum, which is usually assigned to N-deuterated peptide groups in an unordered structure, since SBTI is known to be devoid of alfa-helix by CD and X-ray crystallographic studies. In addition, shoulders are evident at 1632 cm**-1 and 1676 cm**-1, which correspond probably to the v(PI,0) and v(O,pi) components assigned to an antiparallel chain ß-pleated sheet structure. Parameters (maximum absorptivity, wavenumber at the maximum of the band, and half-width at the band at half-height) for the four Gaussian component bands (in which the amide I' band was resolved) are given. A crude estimation of 4% is obtained for antiparallel ß-sheet in SBTI, i.e., this protein would be practically devoid of such a ß-structure. Notwithstanding the fact that this results is apparently in agreement with the far-UV CD spectrum (data reported in the literature), the predominant conformation class found in SBTI has been demonstrated to be approximate ß-sheet structures, with a small amount of regular sheet

Trypsin and chymotryosin inhinitor of Vigna unguiculata seeds - involvement of tyrosyls in its interaction with proteases

When trypsin and chymotrypsin inhibitor of Vigna unguiculata seeds (black-eyed pea trypsin and chymotrypsin inhibitor, BTCI) combines with ß-trysin, 4.0, 1.5, 5.0, 5.8, and 6.6 tyrosyl residues are shield from reaction with N-acetylimidazole, at reagent/protein molar ratios of 60, 120, 200, 350 and 500, respectively. This may result from the presence of tyrosyl residues in the zone of contact between enzyme and inhibitor. In the interaction of BTCI and alpha-chymotrypsin, only 0.6 tyrosyl residues are shielded from the reaction with N-acetylimidazole, at a 500-fold reagent molar excess