Structure-activity relationships derived by machine learning: the use of atoms and their bond connectivities to predict mutagenicity by inductive logic programming.

We present a general approach to forming structure-activity relationships (SARs). This approach is based on representing chemical structure by atoms and their bond connectivities in combination with the inductive logic programming (ILP) algorithm PROGOL. Existing SAR methods describe chemical structure by using attributes which are general properties of an object. It is not possible to map chemical structure directly to attribute-based descriptions, as such descriptions have no internal organization. A more natural and general way to describe chemical structure is to use a relational description, where the internal construction of the description maps that of the object described. Our atom and bond connectivities representation is a relational description. ILP algorithms can form SARs with relational descriptions. We have tested the relational approach by investigating the SARs of 230 aromatic and heteroaromatic nitro compounds. These compounds had been split previously into two subsets, 188 compounds that were amenable to regression and 42 that were not. For the 188 compounds, a SAR was found that was as accurate as the best statistical or neural network-generated SARs. The PROGOL SAR has the advantages that it did not need the use of any indicator variables handcrafted by an expert, and the generated rules were easily comprehensible. For the 42 compounds, PROGOL formed a SAR that was significantly (P < 0.025) more accurate than linear regression, quadratic regression, and back-propagation. This SAR is based on an automatically generated structural alert for mutagenicity.

Ethyl-substituted erythromycin derivatives produced by directed metabolic engineering

A previously unknown chemical structure, 6-desmethyl-6-ethylerythromycin A (6-ethylErA), was produced through directed genetic manipulation of the erythromycin (Er)-producing organism Saccharopolyspora erythraea. In an attempt to replace the methyl side chain at the C-6 position of the Er polyketide backbone with an ethyl moiety, the methylmalonate-specific acyltransferase (AT) domain of the Er polyketide synthase was replaced with an ethylmalonate-specific AT domain from the polyketide synthase involved in the synthesis of the 16-member macrolide niddamycin. The genetically altered strain was found to produce ErA, however, and not the ethyl-substituted derivative. When the strain was provided with precursors of ethylmalonate, a small quantity of a macrolide with the mass of 6-ethylErA was produced in addition to ErA. Because substrate for the heterologous AT seemed to be limiting, crotonyl-CoA reductase, a primary metabolic enzyme involved in butyryl-CoA production in streptomycetes, was expressed in the strain. The primary macrolide produced by the reengineered strain was 6-ethylErA.

Effects of unique ion chemistry on thin-film growth by plasma–surface interactions

Plasma processing is a standard industrial method for the modification of material surfaces and the deposition of thin films. Polyatomic ions and neutrals larger than a triatomic play a critical role in plasma-induced surface chemistry, especially in the deposition of polymeric films from fluorocarbon plasmas. In this paper, low energy CF3+ and C3F5+ ions are used to modify a polystyrene surface. Experimental and computational studies are combined to quantify the effect of the unique chemistry and structure of the incident ions on the result of ion-polymer collisions. C3F5+ ions are more effective at growing films than CF3+, both at similar energy/atom of ≈6 eV/atom and similar total kinetic energies of 25 and 50 eV. The composition of the films grown experimentally also varies with both the structure and kinetic energy of the incident ion. Both C3F5+ and CF3+ should be thought of as covalently bound polyatomic precursors or fragments that can react and become incorporated within the polystyrene surface, rather than merely donating F atoms. The size and structure of the ions affect polymer film formation via differing chemical structure, reactivity, sticking probabilities, and energy transfer to the surface. The different reactivity of these two ions with the polymer surface supports the argument that larger species contribute to the deposition of polymeric films from fluorocarbon plasmas. These results indicate that complete understanding and accurate computer modeling of plasma–surface modification requires accurate measurement of the identities, number densities, and kinetic energies of higher mass ions and energetic neutrals.

Synthesis and spectroscopic characterization of site-specific 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine oligodeoxyribonucleotide adducts

The aim of the present study is to determine the chemical structure and conformation of DNA adducts formed by incubation of the bioactive form of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), N-acetoxy-PhIP, with a single-stranded 11mer oligodeoxyribonucleotide. Using conditions optimized to give the C8-dG-PhIP adduct as the major product, sufficient material was synthesized for NMR solution structure determination. The NMR data indicate that in duplex DNA this adduct exists in equilibrium between two different conformational states. In the main conformer, the covalently bound PhIP molecule intercalates in the helix, whilst in the minor conformation the PhIP ligand is probably solvent exposed. In addition to the C8-dG-PhIP adduct, at least eight polar adducts are found after reaction of N-acetoxy-PhIP with the oligonucleotide. Three of these were purified for further characterization and shown to exhibit lowest energy UV absorption bands in the range 342–347 nm, confirming the presence of PhIP or PhIP derivative. Accurate mass determination of two of the polar adducts by negative ion MALDI-TOF MS revealed ions consistent with a spirobisguanidino-PhIP derivative and a ring-opened adduct. The third adduct, which has the same mass as the C8-dG-PhIP oligonucleotide adduct, may contain PhIP bound to the N2 position of guanine.

A new endogenous natriuretic factor: LLU-alpha.

For over three decades, renal physiology has sought a putative natriuretic hormone (third factor) that might control the body's pool of extracellular fluid, an important determinant in hypertension, congestive heart failure, and cirrhosis. In our search for this hormone, we have isolated several pure natriuretic factors from human uremic urine that would appear, alone or in combination, to mark a cluster of phenomena previously presumed to be that of a single "natriuretic hormone." This paper reports the purification, chemical structure, and total synthesis of the first of these compounds, LLU-alpha, which proved to be 2,7,8-trimethyl-2-(beta-carboxyethyl)-6-hydroxychroman, presumably a metabolite of gamma-tocopherol. Both natural LLU-alpha and synthetic material are identical (except for optical activity) with respect to structure and biological activity. It appears that the natriuretic activity of LLU-alpha is mediated by inhibition of the 70 pS K+ channel in the apical membrane of the thick ascending limb of the kidney.

Modulation of cholecystokinin activity by albumin.

We found that a variety of cholecystokinin (CCK) receptor ligands bind to bovine serum albumin (BSA). This binding was rapid, fully reversible, temperature independent, of low affinity, and specific for BSA; it depended on the concentration of BSA, the chemical structure of the ligand, and the chemical composition of the incubation medium. BSA also decreased the binding of 125I-labeled CCK octapeptide (125I-CCK-8) to CCK receptors on pancreatic acini and membranes but increased the potency with which CCK-8 inhibited binding of 125I-CCK-8. These counterintuitive findings appeared to result from BSA altering the affinities of CCK-8 for different affinity states of the pancreatic CCK receptor. An alternate hypothesis is that BSA increased the efficacy of CCK-8 such that it bound to receptors and also caused biochemical changes in other receptors that reduced their ability to bind 125I-CCK-8. BSA enhanced the ability of CCK-8 to stimulate amylase secretion from pancreatic acini and to cause contraction of dispersed gastric smooth muscle cells. Thus, CCK can bind to BSA, and the BSA-CCK complex has substantially different activities from the free, uncomplexed hormone.

CC-1065 and the duocarmycins: unraveling the keys to a new class of naturally derived DNA alkylating agents.

Key studies defining the DNA alkylation properties and selectivity of a new class of exceptionally potent, naturally occurring antitumor antibiotics including CC-1065, duocarmycin A, and duocarmycin SA are reviewed. Recent studies conducted with synthetic agents containing deep-seated structural changes and the unnatural enantiomers of the natural products and related analogs have defined the structural basis for the sequence-selective alkylation of duplex DNA and fundamental relationships between chemical structure, functional reactivity, and biological properties. The agents undergo a reversible, stereoelectronically controlled adenine-N3 addition to the least substituted carbon of the activated cyclopropane within selected AT-rich sites. The preferential AT-rich non-covalent binding selectivity of the agents within the narrower, deeper AT-rich minor groove and the steric accessibility to the alkylation site that accompanies deep AT-rich minor groove penetration control the sequence-selective DNA alkylation reaction and stabilize the resulting adduct. For the agents that possess sufficient reactivity to alkylate DNA, a direct relationship between chemical or functional stability and biological potency has been defined.

Structural basis for chemical inhibition of human blood coagulation factor Xa

Factor Xa, the converting enzyme of prothrombin to thrombin, has emerged as an alternative (to thrombin) target for drug discovery for thromboembolic diseases. An inhibitor has been synthesized and the crystal structure of the complex between Des[1–44] factor Xa and the inhibitor has been determined by crystallographic methods in two different crystal forms to 2.3- and 2.4-Å resolution. The racemic mixture of inhibitor FX-2212, (2RS)-(3′-amidino-3-biphenylyl)-5-(4-pyridylamino)pentanoic acid, inhibits factor Xa activity by 50% at 272 nM in vitro. The S-isomer of FX-2212 (FX-2212a) was found to bind to the active site of factor Xa in both crystal forms. The biphenylamidine of FX-2212a occupies the S1-pocket, and the pyridine ring makes hydrophobic interactions with the factor Xa aryl-binding site. Several water molecules meditate inhibitor binding to residues in the active site. In contrast to the earlier crystal structures of factor Xa, such as those of apo-Des[1–45] factor Xa and Des[1–44] factor Xa in complex with a naphthyl inhibitor DX-9065a, two epidermal growth factor-like domains of factor Xa are well ordered in both our crystal forms as well as the region between the two domains, which recently was found to be the binding site of the effector cell protease receptor-1. This structure provides a basis for designing next generation inhibitors of factor Xa.

Crystal structure of 2,5-diketo-d-gluconic acid reductase A complexed with NADPH at 2.1-Å resolution

The three-dimensional structure of Corynebacterium 2,5-diketo-d-gluconic acid reductase A (2,5-DKGR A; EC 1.1.1.-), in complex with cofactor NADPH, has been solved by using x-ray crystallographic data to 2.1-Å resolution. This enzyme catalyzes stereospecific reduction of 2,5-diketo-d-gluconate (2,5-DKG) to 2-keto-l-gulonate. Thus the three-dimensional structure has now been solved for a prokaryotic example of the aldo–keto reductase superfamily. The details of the binding of the NADPH cofactor help to explain why 2,5-DKGR exhibits lower binding affinity for cofactor than the related human aldose reductase does. Furthermore, changes in the local loop structure near the cofactor suggest that 2,5-DKGR will not exhibit the biphasic cofactor binding characteristics observed in aldose reductase. Although the crystal structure does not include substrate, the two ordered water molecules present within the substrate-binding pocket are postulated to provide positional landmarks for the substrate 5-keto and 4-hydroxyl groups. The structural basis for several previously described active-site mutants of 2,5-DKGR A is also proposed. Recent research efforts have described a novel approach to the synthesis of l-ascorbate (vitamin C) by using a genetically engineered microorganism that is capable of synthesizing 2,5-DKG from glucose and subsequently is transformed with the gene for 2,5-DKGR. These modifications create a microorganism capable of direct production of 2-keto-l-gulonate from d-glucose, and the gulonate can subsequently be converted into vitamin C. In economic terms, vitamin C is the single most important specialty chemical manufactured in the world. Understanding the structural determinants of specificity, catalysis, and stability for 2,5-DKGR A is of substantial commercial interest.

Crystal structure of chemically synthesized [N33A] stromal cell-derived factor 1α, a potent ligand for the HIV-1 “fusin” coreceptor

Stromal cell-derived factor-1α (SDF-1α ) is a member of the chemokine superfamily and functions as a growth factor and chemoattractant through activation of CXCR4/LESTR/Fusin, a G protein-coupled receptor. This receptor also functions as a coreceptor for T-tropic syncytium-inducing strains of HIV-1. SDF-1α antagonizes infectivity of these strains by competing with gp120 for binding to the receptor. The crystal structure of a variant SDF-1α ([N33A]SDF-1α ) prepared by total chemical synthesis has been refined to 2.2-Å resolution. Although SDF-1α adopts a typical chemokine β-β-β-α topology, the packing of the α-helix against the β-sheet is strikingly different. Comparison of SDF-1α with other chemokine structures confirms the hypothesis that SDF-1α may be either an ancestral protein from which all other chemokines evolved or the chemokine that is the least divergent from a primordial chemokine. The structure of SDF-1α reveals a positively charged surface ideal for binding to the negatively charged extracellular loops of the CXCR4 HIV-1 coreceptor. This ionic complementarity is likely to promote the interaction of the mobile N-terminal segment of SDF-1α with interhelical sites of the receptor, resulting in a biological response.

Rapid chemical kinetic techniques for investigations of neurotransmitter receptors expressed in Xenopus oocytes

Xenopus laevis oocytes have been used extensively during the past decade to express and study neurotransmitter receptors of various origins and subunit composition and also to express and study receptors altered by site-specific mutations. Interpretations of the effects of structural differences on receptor mechanisms were, however, hampered by a lack of rapid chemical reaction techniques suitable for use with oocytes. Here we describe flow and photolysis techniques, with 2-ms and 100-μs time resolution, respectively, for studying neurotransmitter receptors in giant (≈20-μm diameter) patches of oocyte membranes, using muscle and neuronal acetylcholine receptors as examples. With these techniques, we find that the muscle receptor in BC3H1 cells and the same receptor expressed in oocytes have comparable kinetic properties. This finding is in contrast to previous studies and raises questions regarding the interpretations of the many studies of receptors expressed in oocytes in which an insufficient time resolution was available. The results obtained indicate that the rapid reaction techniques described here, in conjunction with the oocyte expression system, will be useful in answering many outstanding questions regarding the structure and function of diverse neurotransmitter receptors.

Aneuploidy correlated 100% with chemical transformation of Chinese hamster cells

Aneuploidy or chromosome imbalance is the most massive genetic abnormality of cancer cells. It used to be considered the cause of cancer when it was discovered more than 100 years ago. Since the discovery of the gene, the aneuploidy hypothesis has lost ground to the hypothesis that mutation of cellular genes causes cancer. According to this hypothesis, cancers are diploid and aneuploidy is secondary or nonessential. Here we reexamine the aneuploidy hypothesis in view of the fact that nearly all solid cancers are aneuploid, that many carcinogens are nongenotoxic, and that mutated genes from cancer cells do not transform diploid human or animal cells. By regrouping the gene pool—as in speciation—aneuploidy inevitably will alter many genetic programs. This genetic revolution can explain the numerous unique properties of cancer cells, such as invasiveness, dedifferentiation, distinct morphology, and specific surface antigens, much better than gene mutation, which is limited by the conservation of the existing chromosome structure. To determine whether aneuploidy is a cause or a consequence of transformation, we have analyzed the chromosomes of Chinese hamster embryo (CHE) cells transformed in vitro. This system allows (i) detection of transformation within 2 months and thus about 5 months sooner than carcinogenesis and (ii) the generation of many more transformants per cost than carcinogenesis. To minimize mutation of cellular genes, we have used nongenotoxic carcinogens. It was found that 44 out of 44 colonies of CHE cells transformed by benz[a]pyrene, methylcholanthrene, dimethylbenzanthracene, and colcemid, or spontaneously were between 50 and 100% aneuploid. Thus, aneuploidy originated with transformation. Two of two chemically transformed colonies tested were tumorigenic 2 months after inoculation into hamsters. The cells of transformed colonies were heterogeneous in chromosome number, consistent with the hypothesis that aneuploidy can perpetually destabilize the chromosome number because it unbalances the elements of the mitotic apparatus. Considering that all 44 transformed colonies analyzed were aneuploid, and the early association between aneuploidy, transformation, and tumorigenicity, we conclude that aneuploidy is the cause rather than a consequence of transformation.

Solution structure and peptide binding studies of the C-terminal Src homology 3-like domain of the diphtheria toxin repressor protein

The diphtheria toxin repressor (DtxR) is the best-characterized member of a family of homologous proteins that regulate iron uptake and virulence gene expression in the Gram-positive bacteria. DtxR contains two domains that are separated by a short, unstructured linker. The N-terminal domain is structurally well-defined and is responsible for Fe2+ binding, dimerization, and DNA binding. The C-terminal domain adopts a fold similar to eukaryotic Src homology 3 domains, but the functional role of the C-terminal domain in repressor activity is unknown. The solution structure of the C-terminal domain, consisting of residues N130-L226 plus a 13-residue N-terminal extension, has been determined by using NMR spectroscopy. Residues before A147 are highly mobile and adopt a random coil conformation, but residues A147-L226 form a single structured domain consisting of five β-strands and three helices arranged into a partially orthogonal, two-sheet β-barrel, similar to the structure observed in the crystalline Co2+ complex of full-length DtxR. Chemical shift perturbation studies demonstrate that a proline-rich peptide corresponding to residues R125-G139 of intact DtxR binds to the C-terminal domain in a pocket formed by residues in β-strands 2, 3, and 5, and helix 3. Binding of the proline-rich peptide by the C-terminal domain of DtxR presents an example of peptide binding by a prokaryotic Src homology 3-like protein. The results of this study, combined with previous x-ray studies of intact DtxR, provide insights into a possible biological function of the C-terminal domain in regulating repressor activity.

Photochemically induced nuclear spin polarization in reaction centers of photosystem II observed by 13C-solid-state NMR reveals a strongly asymmetric electronic structure of the P680.+ primary donor chlorophyll

We report 13C magic angle spinning NMR observation of photochemically induced dynamic nuclear spin polarization (photo- CIDNP) in the reaction center (RC) of photosystem II (PS2). The light-enhanced NMR signals of the natural abundance 13C provide information on the electronic structure of the primary electron donor P680 (chlorophyll a molecules absorbing around 680 nm) and on the pz spin density pattern in its oxidized form, P680⨥. Most centerband signals can be attributed to a single chlorophyll a (Chl a) cofactor that has little interaction with other pigments. The chemical shift anisotropy of the most intense signals is characteristic for aromatic carbon atoms. The data reveal a pronounced asymmetry of the electronic spin density distribution within the P680⨥. PS2 shows only a single broad and intense emissive signal, which is assigned to both the C-10 and C-15 methine carbon atoms. The spin density appears shifted toward ring III. This shift is remarkable, because, for monomeric Chl a radical cations in solution, the region of highest spin density is around ring II. It leads to a first hypothesis as to how the planet can provide itself with the chemical potential to split water and generate an oxygen atmosphere using the Chl a macroaromatic cycle. A local electrostatic field close to ring III can polarize the electronic charge and associated spin density and increase the redox potential of P680 by stabilizing the highest occupied molecular orbital, without a major change of color. This field could be produced, e.g., by protonation of the keto group of ring V. Finally, the radical cation electronic structure in PS2 is different from that in the bacterial RC, which shows at least four emissive centerbands, indicating a symmetric spin density distribution over the entire bacteriochlorophyll macrocycle.

Structure of the recombinant full-length hamster prion protein PrP(29–231): The N terminus is highly flexible

The prion diseases seem to be caused by a conformational change of the prion protein (PrP) from the benign cellular form PrPC to the infectious scrapie form PrPSc; thus, detailed information about PrP structure may provide essential insights into the mechanism by which these diseases develop. In this study, the secondary structure of the recombinant Syrian hamster PrP of residues 29–231 [PrP(29–231)] is investigated by multidimensional heteronuclear NMR. Chemical shift index analysis and nuclear Overhauser effect data show that PrP(29–231) contains three helices and possibly one short β-strand. Most striking is the random-coil nature of chemical shifts for residues 30–124 in the full-length PrP. Although the secondary structure elements are similar to those found in mouse PrP fragment PrP(121–231), the secondary structure boundaries of PrP(29–231) are different from those in mouse PrP(121–231) but similar to those found in the structure of Syrian hamster PrP(90–231). Comparison of resonance assignments of PrP(29–231) and PrP(90–231) indicates that there may be transient interactions between the additional residues and the structured core. Backbone dynamics studies done by using the heteronuclear [1H]-15N nuclear Overhauser effect indicate that almost half of PrP(29–231), residues 29–124, is highly flexible. This plastic region could feature in the conversion of PrPC to PrPSc by template-assisted formation of β-structure.