Trypanosoma cruzi, a protozoan parasite, is the causative agent of Chagas disease, a major cause of cardiovascular disease in many Latin American countries. There is an urgent need to develop an improved therapy due to the toxicity of existing drugs and emerging drug resistance. Cruzain, the primary cysteine protease of T cruzi, is essential for the survival of the parasite in host cells and therefore is an important target for the development of inhibitors as potential therapeutics. A novel series of alpha-ketoamide-, alpha-ketoacid-, alpha-ketoester-, and aldehyde-based inhibitors of cruzain has been developed. The inhibitors were identified by screening protease targeted small molecule libraries and systematically optimizing the P1, P2, P3, and P1 ' residues using specific structure-guided methods. A total of 20 compounds displayed picomolar potency in in vitro assays and three inhibitors representing different alpha-keto-based inhibitor scaffolds demonstrated anti-trypanosomal activity in cell culture. A 2.3 angstrom crystallographic structure of cruzain bound with one of the alpha-ketoester analogs is also reported. The structure and kinetic assay data illustrate the covalent binding, reversible inhibition mechanism of the inhibitor. Information on the compounds reported here will be useful in the development of new lead compounds as potential therapeutic agents for the treatment of Chagas disease and as biological probes to study the role that cruzain plays in the pathology. This study also demonstrates the validity of structure-guided approaches to focused library design and lead compound optimization. (c) 2005 Elsevier Ltd. All rights reserved.

Rapid chemical quench methods coupled with off-line detection have proven to be very useful in identifying enzyme reaction intermediates. However, a limitation to this approach involves enzyme intermediates which are too labile under the chemical quenching conditions to allow detection and characterization. In this report, we describe the development of a novel approach for the detection and characterization of enzyme intermediates on the subsecond time scale using a "pulsed flow" method which employs a direct interface between a rapid-mixing device and electrospray ionization mass spectrometry. The application of this technique with the enzyme 5-enolpyruvoyl-shikimate-3-phosphate (EPSP) synthase is demonstrated. This enzyme converts shikimate-3-phosphate (S3P) and phosphoenol pyruvate (PEP) to EPSP and inorganic phosphate. Previous rapid chemical quench studies have shown that this reaction proceeds through a tetrahedral intermediate [Anderson, K. S., et al. (1988) J. Am.Chem. Soc. 110, 6577-6579] formed transiently at the enzyme active site. We have shown that this tetrahedral intermediate can be directly detected on a subsecond time scale without chemical quenching by interfacing a rapid mixing apparatus directly with an on-line electrospray ionization ion trap mass spectrometer. Negative ion mass spectra collected by electrospray ionization indicate peaks for S3P (m/z 253), PEP (m/z 167), EPSP (m/z323), and the tetrahedral intermediate (m/z 421). Further confirmation was provided by performing the same experiment with [13C-1]-labeled PEP. These spectra confirmed the anticipated shift of 1 atomic mass unit for PEP (m/z 168), EPSP (m/z 324), and the tetrahedral intermediate (m/z 422) with no change in S3P (m/z 253). The collision-induced dissociation of the unlabeled tetrahedral intermediate peak (m/z421) produced a daughter ion at m/z 323, which is most likely EPSP resulting from the loss of phosphate and is consistent with previous studies which have examined the chemical breakdown of the tetrahedral intermediate in solution [Anderson, K. S., et al. (1990) J. Biol. Chem. 265, 5567-6672]. This technique is under development and should be a useful method to study the transient formation of enzyme intermediates.

The average structural and dynamic properties of tetragonal hen egg-white lysozyme have been compared, in structures refined at 1.9 A resolution, using data collected at 100 K and 298 K. The molecule expands by 1.8% over this temperature range with the expansion occurring primarily in its small sub-atomic-sized spaces in an anisotropic manner. Hen egg-white lysozyme consists of two domains: domain 1 (residues 40 to 88) is composed primarily of beta-sheet and is observed to expand by only 0.3%; domain 2 (residues 1 to 39 and 89 to 129) is chiefly alpha-helix and is observed to expand by 2.2%. This is consistent with previous observations that proteins composed primarily of alpha-helix expand more with temperature than do those composed primarily of beta-sheet. The largest movement in the molecule is undergone by the two domains of the structure that move further apart as the temperature is raised. This motion is not a cleft opening but rather consists of a tilt by 2.3 degrees of domain 1 away from domain 2. Within the individual domains the largest movement is undergone by loop T1 of domain 2, consisting of residues 17 to 23. This loop moves in the opposite direction to the rest of the molecule as the temperature is raised. Average temperature factors for the room-temperature and low-temperature structures are 15.2 A2 and 8.1 A2, respectively, when all protein atoms are considered, while these values are 14.0 A2 and 7.8 A2, when only main-chain atoms (N, C alpha, C) are taken into account. An examination of the main-chain averaged B-factor per residue shows that residues involved in intermolecular protein-protein contacts, with symmetry-related molecules, have somewhat lower B-factors than the average and undergo smaller than average changes in B-factor as the temperature is lowered.

Two crystals of monoclinic hen egg-white lysozyme were irradiated in a monochromatic synchrotron X-ray beam (lambda = 1.488 angstrom), the first as a rapidly frozen crystal mounted at the end of a glass fiber at low temperature (120 K) and the second mounted in a capillary tube at ambient temperature (298 K). Comparison of oscillation photographs, extending in resolution to 1.85 angstrom and taken from both crystals at zero time and again after a period of exposure in the synchrotron beam (60 min exposure at 120 K; 8 min at 298 K), reveals that radiation-induced decay is not observed at 120 K but is observed, particularly at high resolution, at 298 K. In a separate set of experiments, data sets to 1.9 angstrom resolution at 100 and 298 K were collected from two monoclinic and two tetragonal hen egg-white lysozyme crystals using a rotating-anode source (lambda = 1.5418 angstrom). Before inclusion of solvent molecules, the monoclinic and tetragonal structures at low temperature, where data were collected from rapidly frozen crystals, refined to R = 27.5 and 25.2%, respectively. The structures at ambient temperature, however, where crystals were mounted in capillary tubes, refined to significantly lower values of R = 20.9 and 20.6%. After inclusion of solvent, the R values at convergence were 20.3 and 17.6% for the monoclinic and tetragonal low-temperature structures and 17.9 and 16.2% for the room-temperature structures. Approximately twice the number of water molecules were included in the low-temperature structures at convergence (406 and 237) than could be assigned in the room-temperature structures (191 and 100). These results suggest that data sets from rapidly frozen crystals might generally be expected to yield higher initial R factors, compared to similar room-temperature structures, but that this difference should diminish appreciably as ordered solvent is included in the model. Apart from the general reduction in atomic temperature factors, the enhancement in resolution observed in diffraction patterns obtained from rapidly frozen crystals is probably due, to some significant degree, to the increase in the ordered-solvent content of the low-temperature structures.

The free energy perturbation method has been employed to determine the binding free energy contributions of different groups of two classes of HIV-1 proteinase inhibitors: (1) a hydroxyethylene isostere inhibitor, Ala-Ala-Phe[CH(OH)-CH2]Gly-Val-Val-OMe (reported by Dreyer et al. 1), and a reduced peptide inhibitor, MVT-101 (reported by Miller et al. 2). For the first inhibitor, the configuration of the central hydroxyl group is changed from S to R in two steps. In the first step, the hydroxyl group in the S configuration was mutated to a hydrogen, and in the second step, the hydroxyl group of the (R)-OH analogue of the inhibitor was mutated to a hydrogen. In this way the binding contributions of the hydroxyl group in the two diastereomers are determined separately in addition to obtaining the effect of changing the hydroxyl group configuration from S to R. The calculated free energy difference between the binding of the two diastereomers is 3.37 +/- 0.64 kcal/mol, which is close the experimental value of 2.6 kcal/mol. The calculations on the substitution of Gly by Nle at the P'1 position of the same inhibitor predict an enhancement in the binding by about 1.7 kcal/mol. Similar calculations on the substitution of Nle with Met in MVT-101 inhibitor predict a decrease in binding by about 0.7 kcal/mol. The details of these results will be discussed and compared with the results of a similar study on pepstatin-rhizopus pepsin complex.

Structures using X-ray diffraction data collected to 1.5-A resolution have been determined for the protein ribonuclease-A at nine different temperatures ranging from 98 to 320 K. It is determined that the protein molecule expands slightly (0.4% per 100 K) with increasing temperature and that this expansion is linear. The expansion is due primarily to subtle repacking of the molecule, with exposed and mobile loop regions exhibiting the largest movements. Individual atomic Debye-Waller factors exhibit predominantly biphasic behavior, with a small positive slope at low temperatures and a larger positive slope at higher temperatures. The break in this curve occurs at a characteristic temperature of 180-200 K, perhaps indicative of fundamental changes in the dynamical structure of the surrounding protein solvent. The distribution of protein Debye-Waller factors is observed to broaden as well as shift to higher values as the temperature is increased.

Xenon gas can be used as a heavy atom for determining phases in a protein. We demonstrate that an interpretable electron density map can be obtained for sperm whale metmyoglobin from a single xenon derivative using iterative single isomorphous replacement with the anomalous scattering method.

Certain precursor proteins (APP751 and APP770) of the amyloid beta-protein (AP) present in Alzheimer's disease contain a Kunitz-type serine protease inhibitor domain (APPI). In this study, the domain is obtained as a functional inhibitor through both recombinant (APPIr) and synthetic (APPIs) methodologies, and the solution structure of APPI is determined by 1H 2D NMR techniques. Complete sequence-specific resonance assignments (except for P13 and G37 NH) for both APPIr and APPIs are achieved using standard procedures. Ambiguities arising from degeneracies in the NMR resonances are resolved by varying sample conditions. Qualitative interpretation of short- and long-range NOEs reveals secondary structural features similar to those extensively documented by NMR for bovine pancreatic trypsin inhibitor (BPTI). A more rigorous interpretation of the NOESY spectra yields NOE-derived interresidue distance restraints which are used in conjunction with dynamic simulated annealing to generate a family of APPI structures. Within this family, the beta-sheet and helical regions are in good agreement with the crystal structure of BPTI, whereas portions of the protease-binding loops deviate from those in BPTI. These deviations are consistent with those recently described in the crystal structure of APPI (Hynes et al., 1990). Also supported in the NMR study is the hydrophobic patch in the protease-binding domain created by side chain-side chain NOE contacts between M17 and F34. In addition, the NMR spectra indicate that the rotation of the W21 ring in APPI is hindered, unlike Y21 in BPTI, showing a greater than 90% preference for one orientation in the hydrophobic groove.

Electrophilic catalysis by histidine-95 in triosephosphate isomerase has been probed by using Fourier transform infrared spectroscopy and X-ray crystallography. The carbonyl stretching frequency of dihydroxyacetone phosphate bound to the wild-type enzyme is known to be 19 cm-1 lower (at 1713 cm-1) than that of dihydroxyacetone phosphate free in solution (at 1732 cm-1), and this decrease in stretching frequency has been ascribed to an enzymic electrophile that polarizes the substrate carbonyl group toward the transition state for the enolization. Infrared spectra of substrate bound to two site-directed mutants of yeast triosephosphate isomerase in which histidine-95 has been changed to glutamine or to asparagine show unperturbed carbonyl stretching frequencies between 1732 and 1742 cm-1. The lack of carbonyl polarization when histidine-95 is removed suggests that histidine-95 is indeed the catalytic electrophile, at least for dihydroxyacetone phosphate. Kinetic studies of the glutamine mutant (H95Q) have shown that the enzyme follows a subtly different mechanism of proton transfers involving only a single acid-base catalytic group. These findings suggest an additional role for histidine-95 as a general acid-base catalyst in the wild-type enzyme. The X-ray crystal structure of the H95Q mutant with an intermediate analogue, phosphoglycolohydroxamate, bound at the active site has been solved to 2.8-A resolution, and this structure clearly implicates glutamate-165, the catalytic base in the wild-type isomerase, as the sole acid-base catalyst for the mutant enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

A 96 picosecond dynamics trajectory of myoglobin with five xenon-probe ligands in internal cavities is examined to study the effect of protein motions on ligand motion and internal cavity fluctuations. Average structural and energetic properties indicate that the simulation is well behaved. The average protein volume is similar to the volume of the X-ray model and the main-chain atom root-mean-square deviation between the X-ray model and the average dynamical structure is 1.25 Å. The protein volume oscillates 3 to 4% around the volume of the X-ray structure. These fluctuations lead to changes in the internal free volume and in the size, shape and location of atom-sized cavity features. Transient cavities produced in the simulation have a crucial role in the movement of two of the ligands. One of the ligands escapes to the protein surface, whilst a second ligand travels through the protein interior. Complex gating processes involving several protein residues are responsible for producing the necessary pores through which the ligand passes between transient cavities or packing defects.

A structure of sperm whale metmyoglobin under a nitrogen gas pressure of 145 atm (2200 psi) has been solved by X-ray diffraction using data to 2.0-A resolution. The perturbation of the gas pressure on the overall structure of the protein is minimal with a root mean square deviation of backbone atoms between the pressurized and unpressurized structures of 0.22 A. Additional electron density is observed, however, in two cavities of the protein molecule. The density is interpreted as a nitrogen molecule bound in the proximal cavity and as a water molecule hydrogen bonded in a separate cavity (cavity 3). In addition, alternate conformations are observed for three internal residues (Leu-135, Phe-138, and Ile-142) that border these cavities. These alternate conformations are not observed in atmospheric pressure structures and are presumed due to the effects of pressure and/or gas binding. The appearance of these alternate conformations implies a repacking of the protein interior and produces a new distribution of cavity spaces. The profile of the Debye-Waller factors for the pressurized structure is similar to that for the room pressure except for a small increase in the distal region (residues 61-69) of the protein.

X-ray data have been collected from crystals of bovine pancreatic ribonuclease A at temperatures of 220K and below, by use of a nitrogen-gas cold stream and a computer-controlled diffractometer. One data set, collected from a crystal mounted in the traditional manner in a quartz capillary tube, showed radiation damage of 21% after 14.6 days of exposure to Cu Kα radiation at 160K. Another crystal, mounted on a glass fiber by use of uncured epoxy cement, showed only a 3% decay of the intensity standards after 28.3 days of exposure to the same radiation and at similar temperatures.

The thermal expansion of a protein, metmyoglobin, was investigated by analysis of the refined X-ray crystal structures at 80 and 255-300 K. On heating from 80 to 300 K, the volume occupied by myoglobin increases by approximately 3%. The linear thermal expansion coefficient is estimated to be 115 X 10(-6) K-1. This value is more than twice as large as that of liquid water but less than that of benzene. As the temperature is raised, the internal volume change does not come from the large, atom-sized internal cavities in the structure but from an increase in the small, subatomic free volumes between atoms. The largest expansion occurs in the region of the CD and GH corners; both these regions move away from the center of the protein. The remainder of the expansion results from the lengthening of contacts between segments of secondary structure.

Computational studies are used to investigate the energies of xenon binding to myoglobin and to describe pathways through the protein interior for a metmyoglobin-xenon complex. Empirical energy calculations indicate a favorable enthalpic contribution of 0.6 to 4.2 kcal/mol to xenon binding for four experimentally determined xenon sites. These calculated enthalpies help to explain the different xenon occupancies observed experimentally. A fifth site, modeled in place of the iron co-ordinated water molecule in the distal cavity, is also predicted to bind xenon. The largest contribution to the binding energy is from van der Waals' interactions with smaller contributions from polarization and protein strain terms. Ligand trajectory calculations as well as a new geometric algorithm define a connecting network of channel-like pathways through the static protein structure. One or two pathways appear to lead most easily from each major internal cavity to the protein surface. The importance of these channels in protein dynamics and their implications as routes for ligand motion are discussed.

Chemischer InformationsdienstVolume 16, Issue 23 Physical Organic Chemistry ChemInform Abstract: NEW TECHNIQUE FOR MEASURING CIRCULAR DICHROISM CHANGES ON A NANOSECOND TIME SCALE. APPLICATION TO (CARBONMONOXY)MYOGLOBIN AND (CARBONMONOXY)HEMOGLOBIN J. W. LEWIS, J. W. LEWISSearch for more papers by this authorR. F. TILTON, R. F. TILTONSearch for more papers by this authorC. M. EINTERZ, C. M. EINTERZSearch for more papers by this authorS. J. MILDER, S. J. MILDERSearch for more papers by this authorI. D. KUNTZ, I. D. KUNTZSearch for more papers by this authorD. S. KLIGER, D. S. KLIGERSearch for more papers by this author J. W. LEWIS, J. W. LEWISSearch for more papers by this authorR. F. TILTON, R. F. TILTONSearch for more papers by this authorC. M. EINTERZ, C. M. EINTERZSearch for more papers by this authorS. J. MILDER, S. J. MILDERSearch for more papers by this authorI. D. KUNTZ, I. D. KUNTZSearch for more papers by this authorD. S. KLIGER, D. S. KLIGERSearch for more papers by this author First published: June 11, 1985 https://doi.org/10.1002/chin.198523051AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume16, Issue23June 11, 1985 RelatedInformation

X-ray crystallographic data to 1.9-A resolution were collected on sperm whale metmyoglobin equilibrated with 7 atm of xenon gas. The results indicate four xenon sites of occupancy from 0.45 to 1.0. These sites are located in interior spaces or packing defects of the myoglobin molecule. The effects of the bound xenon on the protein structure are minor, and we observe a small overall reduction in refined isotropic atomic protein temperature factors. We interpret the results as a confirmation that, on a time-averaged basis, cavities exist within the myoglobin molecule and suggest that the binding of small ligands in these cavities affects the internal motions and conformational substrates of the protein.

Nuclear magnetic resonance studies of 129Xe are consistent with one kinetically distinguishable binding environment in methemoglobin and two in metmyoglobin. The Xe binding site in methemoglobin is assigned to a cavity formed by the A-B and G-H corners of the globin chain [Schoenborn, B.P. (1965) Nature (London) 208, 760-762]. The small differences between alpha-hemoglobin and beta-hemoglobin are not resolved by the NMR experiments. The Xe association rate constant at 18 degrees C with methemoglobin is greater than 6 X 10(7) M-1 s-1 with an activation barrier of approximately 13 kcal/mol. One of the binding sites in metmyoglobin associated with a cavity on the proximal side of the porphyrin ring, opposite the O2 binding site [Schoenborn, B.P., Watson, H.C., & Kendrew, J.C. (1965) Nature (London) 207, 28-30]. An estimate of the association rate constant of Xe at 18 degrees C is 1 X 10(7) M-1 s-1 with an activation barrier of approximately 16 kcal/mol. The second metmyoglobin binding site has similar NMR and kinetic properties of those for methemoglobin.