Compound acquisition strategies.

Compound acquisition has always been a very important component in the discovery and development of new biologically active entities. With the rapid advances in high throughput screening coupled with the ever-decreasing time requirements for the discovery phase, the number and quality of compounds screened is of great importance. This presentation will discuss some of the techniques and processes that can be used in compound acquisition.

Cosmic radiation and magnetic field exposure to airline flight crews.

BACKGROUND: Flight crews are exposed to elevated levels of cosmic radiation and to magnetic fields generated by the aircraft's electrical system. The purpose of this study was to quantify these two occupational exposures. METHODS: Magnetic fields were measured during 37 flights (23 in the cockpit and 14 in the cabin) using an Emdex Lite personal dosimeter. All cockpit measurements were taken on the B737/200. Cabin measurements were taken in several aircraft types, including the B737, B757, DC9, and L1011. Cosmic radiation was computer estimated for 206 flights using the Federal Aviation Administration's program CARI-3C. RESULTS: Magnetic field levels in the cockpit had a mean value of approximately 17 milliGauss (mG), while cabin measurements were lower (mean values of approximately 3 or less in economy, 6 in first class, 8 in front serving areas). Cosmic radiation equivalent dose rates to bone marrow and skeletal tissue ranged from 0.3 to 5.7 microsieverts per hour. CONCLUSIONS: Elevated magnetic field levels in front serving areas and the cockpit suggest the need for further study to evaluate long-term exposure to flight crew members who work in these areas. Cosmic radiation levels are well below occupational limits for adults, but may require some pregnant flight crew members to adjust their flying time or routes.

Mortality among US commercial pilots and navigators.

The airline industry may be an occupational setting with specific health risks. Two environmental agents to which flight crews are known to be exposed are cosmic radiation and magnetic fields generated by the aircraft's electrical system. Other factors to be considered are circadian disruption and conditions specific to air travel, such as noise, vibration, mild hypoxia, reduced atmospheric pressure, low humidity, and air quality. This study investigated mortality among US commercial pilots and navigators, using proportional mortality ratios for cancer and noncancer end points. Proportional cancer mortality ratios and mortality odds ratios were also calculated for comparison to the proportional mortality ratios for cancer causes of death. Results indicated that US pilots and navigators have experienced significantly increased mortality due to cancer of the kidney and renal pelvis, motor neuron disease, and external causes. In addition, increased mortality due to prostate cancer, brain cancer, colon cancer, and cancer of the lip, buccal cavity, and pharynx was suggested. Mortality was significantly decreased for 11 causes. To determine if these health outcomes are related to occupational exposures, it will be necessary to quantify each exposure separately, to study the potential synergy of effects, and to couple this information with disease data on an individual basis.

Designing inhibitors of the metalloproteinase superfamily: comparative analysis of representative structures.

Structural comparisons of representative members of the zinc metalloproteinase superfamily show that the key secondary structural elements are conserved, in spite of major variations in the sequences including insertions and deletions of functional domains. Major differences between the matrix metalloproteinases (matrixins) are clustered in two regions forming the entrance to the active site and hence may be determinants of substrate selectivity. A comparison of the structures of matrixin-inhibitor complexes shows that there are significant differences even among the closely related matrixins, not only in the peripheral regions but also in the specificity pockets; these differences offer an excellent opportunity for the design of specific inhibitors targetted to individual members.

X-ray structure of a hydroxamate inhibitor complex of stromelysin catalytic domain and its comparison with members of the zinc metalloproteinase superfamily.

BACKGROUND: Stromelysin belongs to a family of zinc-dependent endopeptidases referred to as matrix metalloproteinases (MMPs, matrixins) because of their capacity for selective degradation of various components of the extracellular matrix. Matrixins play key roles in diseases as diverse as arthritis and cancer and hence are important targets for therapeutic intervention. RESULTS: The crystal structure of the stromelysin catalytic domain (SCD) with bound hydroxamate inhibitor, solved by multiple isomorphous replacement, shows deep S1' specificity pocket which explains differences in inhibitors binding between the collagenases and stromelysin. The binding of calcium ions by loops at the two ends of a beta-strand which marks the boundary of the active site provides a structural rationale for the importance of these cations for stability and catalytic activity. Major differences between the matrixins are clustered in two regions forming the entrance to the active site and hence may be determinants of substrate selectivity. CONCLUSIONS: Structural comparisons of SCD with representative members of the metalloproteinase superfamily clearly highlight the conservation of key secondary structural elements, in spite of major variations in the sequences including insertions and deletions of functional domains. However, the three-dimensional structure of SCD, which is generally closely related to the collagenases, shows significant differences not only in the peripheral regions but also in the specificity pockets; these latter differences should facilitate the rational design of specific inhibitors.

Enhancing the diversity of a corporate database using chemical database clustering and analysis.

The contribution that the Chemical Abstracts structural database (CAST-3D) and the Maybridge database (MAY) would make to diversifying the structural information and property space spanned by our corporate database (CBI) is assessed. A subset of the CAST-3D database has been selected to augment the structural diversity of various electronic databases used in computer-assisted drug design projects. The analysis of the MAY database directly offers the potential to expand the CBI compound library, but also provides a source for structural diversity in a format suitable for computer-assisted database searching and molecular design. The analysis performed is twofold. First, a nonhierarchical clustering technique available in the Daylight clustering package is applied to evaluate the structural differences between databases. The comparison is then extended to analyze various structure-derived property spaces calculated from molecular descriptors such as the logarithm of the octanol-water partition coefficient (CLOGP), the molar refractivity (CMR) and the electronic dipole moment (CDM). The diversity contribution of each database to these property spaces is quantified in relation to our corporate database.

Characterization of endothelins as chemoattractants for human neutrophils.

The chemotactic response of human neutrophils to endothelins (ET) and ET-derived peptides was examined. ET-1, ET-2, and ET-3 elicited maximum responses at 10(-7), 3.3 x 10(-8) and 10(-7) M, respectively. Relative activities of the peptides at their optimal concentrations were: ET-1, ET-2 > ET-3. The chemotactic activity of ET-1 was localized to its Leu6-Met7-Asp8 segment. Conformation of the disulfide-linked Cys3-Cys11 loop appears to be critical for proper orientation of the chemotactic epitope. In comparison, ET-1 failed to stimulate the neutrophil respiratory burst, degranulation or arachidonic acid metabolism. These results demonstrate the selective chemoattractant activity of endothelins for human neutrophils.

The solution structure of a cyclic endothelin antagonist, BQ-123, based on 1H-1H and 13C-1H three bond coupling constants.

A cyclic pentapeptide endothelin antagonist, cyclo(dTrp-dAsp-Pro-dVal-Leu), recently reported (K. Ishikawa et al., 13th Am. Pept. Symp., Cambridge MA, 1991) has been studied by NMR spectroscopy and molecular modeling. A stable structure has been determined without the use of nuclear Overhauser effects and is based primarily on homonuclear and heteronuclear three bond coupling constants. The 13C-edited TOCSY experiment is demonstrated at natural abundance and approximately 30 mM peptide concentrations. Three bond 13C-1H coupling constants obtained by this method are shown to reduce the ambiguity in phi angle determination which exists when only interproton coupling constants are used. Three out of four phi angles were determined uniquely by this method and the fourth was reduced to two possible values. The proline phi angle was determined to be -78 degrees based on the 3JH alpha, H beta and 3JH alpha, H beta coupling constants. Comparison of amide proton temperature dependence, chemical shifts and vicinal proton coupling constants in a 20% acetonitrile/80% water solvent mixture and in (CD3)2SO indicates that the structure is similar in both solvents.

Importance of secondary structure for endothelin binding and functional activity.

ET-1[16-Phe] and ET-1[12-Pro] were prepared in order to investigate the importance of secondary structure of ET-1 for receptor binding and function. ET-1[16-Phe] displayed the greatest binding and contractile potency of the ET-analogs tested in rabbit pulmonary artery, rat aorta, and rat left atria. ET-1[12-Pro] also exhibited low nanomolar binding in these tissues but showed less contractile activity than ET-1[16-Phe] or ET-1. The results indicate that the helical region between residues Lys9 and Cys15 of ET-1 is not critical for receptor binding and functional activity. However, replacement of His16 with Phe altered the charge characteristics of the C-terminal region of ET-1 producing the most potent ET-1 analog yet reported.

An investigation of Chromatium vinosum high-potential iron-sulfur protein by EPR and Mossbauer spectroscopy; evidence for a freezing-induced dimerization in NaCl solutions.

The high-potential iron-sulfur protein (HiPIP) from Chromatium vinosum contains a cubane prosthetic group that shuttles between the [4Fe-4S]3+,2+ states. We find that the EPR spectra from this protein can be explained as a sum of two components, a major one with g = 2.02; 2.04; 2.12, and a minor one with g = 2.04; 2.07; approximately 2.13. In the presence of 0.1-2.0 M NaCl, freezing induces polymerization of the protein (presumably dimers), which is detected as intercluster spin-spin interaction in the EPR. The observed spin-spin interactions are interpreted as being due to two very similar dimeric structures in an approx. 1:2 ratio. Computer simulation of the X- and Q-band EPR spectra shows that the z-components of the g-tensors in each dimer pair must be co-linear, with center-to-center distances between the clusters of approximately 13 A and approximately 16 A. Inspection of possible dimeric structures of C. vinosum HiPIP by standard molecular graphics procedures revealed that the Fe/S cluster is exposed toward a flattened surface and is accessible to solvent. Moreover, the Fe/S clusters in two HiPIP molecules can easily achieve a center-to-center distance of approximately 14 A when approaching along a common 3-fold axis that extends through the S4 sulfur atom of the cubane; the z-component of the EPR g-tensor is co-linear with this symmetry axis.

The conformation of endothelin-1 in aqueous solution: NMR-derived constraints combined with distance geometry and molecular dynamics calculations.

The aqueous solution conformation of the bicyclic, 21 amino acid vasoconstrictor peptide, endothelin-1, has been determined using two dimensional NMR and a combination of distance geometry and molecular dynamics. The dominant structural feature is a helical region between Lys9 and Cys15 characterized by strong NHi-NHi+1 NOEs and several long range NOEs spanning 3 to 5 residues. Solvent inaccessibility and possible hydrogen bonding in the Cys3-Cys11 loop is suggested by the temperature independence of the chemical shifts of several amide protons. There is no evidence for association of the C-terminal hexapeptide with the bicyclic region.

Breast cancer screening in a biracial community: the Charleston tricounty experience.

Breast cancer remains a major cause of death among women in South Carolina. Mammography, breast self-examination, and clinical breast examination are effective methods for early detection and subsequent mortality reduction. The Tricounty Breast Cancer Screening Survey assessed knowledge of these methods and recommendations among 503 women in the Charleston area. While 57% of all respondents reported performing breast self-examination at least once per month, 13% of blacks and 6% of whites reported that they do not know how to perform the procedure. Clinical breast examinations within the past year were reported by 69%, yet 11% of blacks and 4% of whites reported that they had never had the examination. More than one third (40%) of all 503 women reported ever having had a mammogram, and 22% reported having had one within the past year. However, 18% of the blacks and 5% of whites reported never having heard of the procedure. The major barriers to mammography appear to be the belief that women do not need regular mammograms and the lack of recommendations by their physicians. Survey results support the need for educating women about what the procedures are, the importance of using them regularly, and the means to comply with them.

Conformational effects of chiral alpha,alpha-dialkyl amino acids. I. C-terminal tetrapeptides of emerimicin containing alpha-ethylalanine.

The syntheses and the crystal structures of the C-terminal tetrapeptide fragments of emerimicin IV and III, Boc-R-EtA-Hyp(Bzl)-Ala-Phol and Boc-R-EtA-Hyp(Bzl)-MeA-Phol, containing the chiral alpha,alpha-dialkyl amino acid, R-alpha-ethylalanine (R-EtA) are reported. The two peptides are isomorphous and assume a 3(10)-helical conformation in the crystal. A comparison of the crystal data on alpha,alpha-dialkyl amino acids indicates that alkyl substituents larger than a methyl group do not preclude peptides containing these amino acids from assuming the conformations associated with minima which have been well characterized for alpha-methylalanine.

Identification of the T-cell and Ia contact residues of a T-cell antigenic epitope.

The precise molecular structure of the antigenic determinant recognized by the T-cell receptor of the CD4-positive cell has not been completely resolved. A major advance in our understanding of this issue has been made by our demonstration of a direct association between an immunogenic peptide and a purified Ia molecule. The most likely and economical hypothesis is that antigen binds directly to an Ia molecule creating the antigenic determinant and that this antigen-Ia complex is recognized by the T-cell receptor. We examined in detail a determinant of hen egg-white lysozyme (HEL) contained in the tryptic fragment HEL(46-61), recognized by T cells in H-2k strains of mice. This peptide binds with a Kd of approximately 3 microM to I-Ak molecules. We have already ascertained that (1) the 10-mer HEL(52-61) is the shortest stimulatory peptide; (2) the Leu56 residue, the only residue different from mouse lysozyme, is responsible for the immunogenicity; (3) the Leu56 and Tyr53 residues are critical for recognition by the T-cell receptor and (4) HEL(46-61) generates multiple determinants when it associated with the I-Ak molecule. If antigen and Ia interact, the antigen must have two features: it must bind to an Ia molecule and also interact with the T-cell receptor. The two sites do not appear to be laterally separable in this peptide and are therefore probably composed of distinct but interspersed amino-acid residues. We have now identified the three residues of HEL(52-61) that contact the T-cell receptor and three other residues that contact the I-Ak molecule. From modelling studies we also propose that HEL(52-61) assumes an alpha-helical conformation as it is bound to I-Ak and recognized by the T-cell receptor.

Calibration of effective van der Waals atomic contact radii for proteins and peptides.

Effective van der Waals radii were calibrated in such a way that molecular models built from standard bond lengths and bond angles reproduced the amino acid conformations observed by crystallography in proteins and peptides. The calibrations were based on the comparison of the Ramachandran plots prepared from high-resolution X-ray data of proteins and peptides with the allowed phi, psi torsional angle space for the dipeptide molecular models. The calibrated radii are useful as criteria with which to filter energetically improbable conformations in molecular modeling studies of proteins and peptides.

Juvenile hypertension: highlights of a workshop.

The workshop was successful in achieving its two major goals: (1) the scientific updating of research issues in pediatric blood pressure determinants and in pediatric hypertension, and (2) the delineation of future research objectives. These objectives are itemized at the end of the workshop proceedings and, in brief, center around the need for better definitions of normal and abnormal blood pressures in youth, the identification of variables in childhood capable of indicating which children are at risk for developing hypertension as adults, and finally, a thoughtful list of additional research issues germane to the explanation of primary and secondary forms of hypertension.