Comparing protein structures: a Gaussian-based approach to the three-dimensional structural similarity of proteins.

This study describes a new method for comparing three-dimensional protein structures based on an optimal alignment of their steric fields. The method is based upon the use of spherical Gaussian functions located on individual atoms. This representation generates a flexible description of the underlying fold geometry of proteins that can be adjusted by changing the 'width' of the Gaussians. Reducing the width sharpens the representation and leads to a more 'atomlike' description; increasing the width creates a fuzzier representation that preserves the general shape features of the chain fold but with a consequent loss in atomic resolution. The width used in this study is based upon the features of individual atoms and provides a representation that is quite robust with respect to the variety of geometric features typically encountered in the alignment process. In addition, a post-alignment analysis is performed that generates sequence alignments from the corresponding structure alignments. An example, based on five mammalian and fungal matrix metalloproteinase crystal structures (human fibroblast collagenase, neutrophil collagenase, stromelysin, astacin, and adamalysin), illustrates a number of features of the Gaussian-based approach.

Identification of novel potent hydroxamic acid inhibitors of peptidyl deformylase and the importance of the hydroxamic acid functionality on inhibition.

Peptidyl deformylase (PDF) is a metallo protease that catalyzes the removal of a formyl group from the N-termini of prokaryotic prepared polypeptides, an essential step in bacterial protein synthesis. Screening of our compound collection using Staphylococcus aureus PDF afforded a very potent inhibitor with an IC(50) in the low nanomolar range. Unfortunately, the compound that contains a hydroxamic acid did not exhibit antibacterial activity (MIC). In order to address the lack of activity in the MIC assay and to determine what portion of the molecule was responsible for binding to PDF, we prepared several analogues. This paper describes our findings that the hydroxamic acid functionality found in 1 is mainly responsible for the high affinity to PDF. In addition, we identified an alternative class of PDF inhibitors, the N-hydroxy urea 18, which has both PDF and antibacterial activity.

A molecular-field-based similarity study of non-nucleoside HIV-1 reverse transcriptase inhibitors. 2. The relationship between alignment solutions obtained from conformationally rigid and flexible matching.

An analysis of the relationship among alignment solutions obtained from field-based matching of a representative set of rigid conformers of three non-nucleoside HIV-1 reverse transcriptase inhibitors and solutions obtained from flexible matching of the same conformers is presented. In some cases, different alignment solutions obtained from rigid matching converge to the same solution when conformational rigidity is relaxed, indicating that a reduced set of conformers per molecule may be sufficient in many field-based similarity studies. Furthermore, the results also indicate the importance of going beyond the pairwise similarity level to obtain consistent solutions in flexible-matching studies. In this respect, the best conformationally flexible multi-molecule alignment obtained is found to be in good agreement with the relative binding geometry and orientation found experimentally from protein-ligand crystal structures. The rms separation between corresponding atoms in computed and 'experimental' sets of three inhibitor structures is 0.94 A.

Field-based similarity forcing in energy minimization and molecular matching.

A new field-based similarity forcing procedure for matching conformationally-flexible molecules is presented. The method extends earlier work on similarity matching of molecules based upon the program MIMIC, by directly coupling a similarity function to a molecular mechanics force field. In this way conformational energetics are fully accounted for in the similarity matching process. Simultaneous similarity/conformational searches can then be undertaken within a Monte Carlo or molecular dynamics framework. Here, a Monte Carlo approach is used to provide a simple example of two HIV-1 reverse transcriptase inhibitors, nevirapine and alpha APA, that illustrates the basic characteristics of the method and suggests areas for further investigation.

A molecular-field-based similarity study of non-nucleoside HIV-1 reverse transcriptase inhibitors.

This article describes a molecular-field-based similarity method for aligning molecules by matching their steric and electrostatic fields and an application of the method to the alignment of three structurally diverse non-nucleoside HIV-1 reverse transcriptase inhibitors. A brief description of the method, as implemented in the program MIMIC, is presented, including a discussion of pairwise and multi-molecule similarity-based matching. The application provides an example that illustrates how relative binding orientations of molecules can be determined in the absence of detailed structural information on their target protein. In the particular system studied here, availability of the X-ray crystal structures of the respective ligand-protein complexes provides a means for constructing an 'experimental model' of the relative binding orientations of the three inhibitors. The experimental model is derived by using MIMIC to align the steric fields of the three protein P66 subunit main chains, producing an overlay with a 1.41 A average rms distance between the corresponding C alpha's in the three chains. The inter-chain residue similarities for the backbone structures show that the main-chain conformations are conserved in the region of the inhibitor-binding site, with the major deviations located primarily in the 'finger' and RNase H regions. The resulting inhibitor structure overlay provides an experimental-based model that can be used to evaluate the quality of the direct a priori inhibitor alignment obtained using MIMIC. It is found that the 'best' pairwise alignments do not always correspond to the experimental model alignments. Therefore, simply combining the best pairwise alignments will not necessarily produce the optimal multi-molecule alignment. However, the best simultaneous three-molecule alignment was found to reproduce the experimental inhibitor alignment model. A pairwise consistency index has been derived which gauges the quality of combining the pairwise alignments and aids in efficiently forming the optimal multi-molecule alignment analysis. Two post-alignment procedures are described that provide information on feature-based and field-based pharmacophoric patterns. The former corresponds to traditional pharmacophore models and is derived from the contribution of individual atoms to the total similarity. The latter is based on molecular regions rather than atoms and is constructed by computing the percent contribution to the similarity of individual points in a regular lattice surrounding the molecules, which when contoured and colored visually depict regions of highly conserved similarity. A discussion of how the information provided by each of the procedures is useful in drug design is also presented.

Synthesis and antibacterial activity of [6,5,5] and [6,6,5] tricyclic fused oxazolidinones.

A series of conformationally restricted, [6,5,5] and [6,6,5] tricyclic fused oxazolidinones were synthesized and tested for antibacterial activity. Several compounds in the trans-[6,5,5] series demonstrated potent in vitro and in vivo activity. This work provides valuable information regarding the preferred conformational orientation of the oxazolidinones at the binding site.

A molecular field-based similarity approach to pharmacophoric pattern recognition.

The use of molecular field-based similarity approaches for obtaining quality molecular alignments and for identifying field-based patterns in bioactive molecules is described. In addition to pairwise similarities, computation of multimolecule similarities affords a means for determining consensus multimolecule alignments. These multimolecule alignments constitute the basis for developing models for the relative binding of bioactive molecules to common protein-binding sites and for the graphical portrayal of molecular field similarity surface plots that identify, visually, molecular regions possessing similar molecular field characteristics. The latter information can then be exploited in the design of molecules that mimic appropriate characteristics of these highly similar steric and electrostatic domains. Regions with low steric and electrostatic similarity in suitably aligned sets of bioactive molecules represent tolerant domains where new structural motifs can be incorporated without significant reductions in activity. To illustrate the potential applicability of the actual molecular field-based similarity approaches to the design of bioactive molecules, a study on a set of HIV-1 protease inhibitors is presented.

Genetically modified PC12 brain grafts: survivability and inducible nerve growth factor expression.

Neural transplantation of genetically modified cells has been successfully employed to reverse functional deficits in animal models of neurodegenerative disorders, including Parkinson's disease. While implanted PC12 cells secrete dopamine in vivo and can ameliorate dopamine deficiency in parkinsonian rat model systems, these cells either degenerate within 2-3 wk postimplantation (presumably due to the lack of neural trophic factor support at the site of implantation), or in some cases, form a tumor mass leading to the death of the host animal. To address these limitations, we have developed a genetically modified PC12 cell line that can synthesize nerve growth factor (NGF) under the control of a zinc-inducible metallothionein promoter. When implanted in the rat striatum and under in vivo zinc stimulation, these cells will neuro-differentiate, express tyrosine hydroxylase, and will undergo survival through potential autocrine trophic support. This regulatable cell line and general approach may provide additional insight on the potential utilization of cell transplants for treatment of Parkinson's disease and other neurodegenerative disorders.

Intraspinal extradural meningeal cyst demonstrating ball-valve mechanism of formation. Case report.

A diverse collection of unverified theories as to the etiology of extradural meningeal cysts have been previously proposed. One case of intraspinal extradural meningeal cyst of the thoracolumbar region is presented in which a ball-valve mechanism involving an idiopathic dural rent and a herniated segment of an underlying dorsal rootlet was suggested by the operative findings. Closure of the dural rent with marsupialization of the meningeal cyst obliterated this extradural lesion. The ball-valve mechanism of formation and other previously proposed theories are discussed.

The effect of 16 beta-substitution on the structure and activity of digitoxigenin: is there an additional binding interaction with Na+,K+-ATPase?

We have studied the basis of the effect of 16 beta-substitution on the structure and activity of digitoxigenin derivatives by examining the crystal structures of these compounds and their inhibitory activity toward the receptor for these drugs, Na+,K+-ATPase. To understand the increase in inhibitory activity of the 16 beta-ester compounds and the decrease in activity of gitoxigenin (16 beta-hydroxydigitoxigenin), both with respect to digitoxigenin, we have compared the observed conformations of gitoxigenin, gitoxigenin 16 beta-formate, and other 16 beta-esters to that of digitoxigenin. Our data do not support the possibility of hydrogen bonding between the 16 beta-hydroxyl of gitoxigenin and the lactone ring, previously suggested to account for the decreased activity of gitoxigenin vis à vis digitoxigenin, but, rather, suggest that the decreased activity may be due to an intramolecular hydrogen bond between the hydroxyls on C-14 and C-16 and an unusual D-ring conformation which combine to alter the carbonyl oxygen of the lactone ring away from the putative active position. In contrast, the 16 beta-ester moiety has a preferred conformation which may serve to fix the lactone ring in the active conformation. Thus, the increased activity of the 16 beta-esters cannot be explained by altered carbonyl oxygen position and may be related to an additional receptor binding site for the ester moiety.

The inotropic activity of digitalis genins is dependent upon C(17) side-group carbonyl oxygen position.

The inotropic potencies of four digitalis genins were studied utilizing cat left atrial strips. The genin concentration required to induce a 50% increase of isometric tension (T50) was found to closely correlate with the degree of displacement (D) of the C(17) side-group carbonyl oxygen from the position of that atom in digitoxigenin. The line of regression was: log T50 = 0.54D - 6.85, r2 = 0.98, P less than 0.008. These observations were related to recently reported cat ventricular Na+, K+ -ATPase inhibitory potencies of the same genins [expressed as 50% inhibitory (I50) concentrations]. I50 correlated strongly with T50: log I50 = 0.78 log T50 - 1.68, r2 = 0.99, P less than 0.003. Thus, the activity of digitalis genins towards their receptor in intact cardiac tissue is closely related to genin carbonyl oxygen position as well as to Na+, K+ -ATPase inhibitory activity. These results further support our earlier conclusions, based upon isolated Na+, K+ -ATPase studies, that the digitalis genin C(17) side-group carbonyl oxygen position versus activity relationship is biologically relevant and may prove to be a useful unifying structural model in the further elucidation of the mechanism of digitalis-receptor interactions.

Positive and electron capture negative ion methane chemical ionization mass spectrometry of pyrrolizidine alkaloids.

Pulsed positive and negative ion methane chemical ionization mass spectrometry of pyrrolizidine alkaloids is reported. Positive ion spectra are characterized by a high relative abundance of [MH]+ ions while the negative ion spectra exhibit ion peaks due to dissociative electron capture. Fragmentation in both positive and negative ion spectra primarily occurs at the ester groups with the positive charge residing with the pyrrolizidine ring system while the negative charge in contrast tends to reside with the necic acid moiety. Esterification at C-9 v. C-7 can be distinguished for non-cyclic esters of retronecine in the positive ion spectra.

Cardiac glycosides. 1. A systematic study of digitoxigenin D-glycosides.

A series of digitoxigenin glycosides was studied: five with beta-D-sugars varying stepwise in sugar structure from beta-D-digitoxose to beta-D-galactose, including one beta-D/alpha-D pair. I50 values for these glycosides and digitoxigenin were determined with hog kidney Na+, K+-ATPase. These data suggest a major and unexpected role for 4'-OH conformation in the sugar. All the glycosides with an equatorial 4'-OH were more active than the two with the 4'-OH axial [digitoxigenin beta-D-galactoside (6) I50 = 6.45 X 10(-8) M; digitoxigenin 2'-deoxy-alpha-D-ribo-hexopyranoside (alpha-3a) I50 = 9.33 X 10(-8) M; digitoxigenin I50 = 1.17 X 10(-7) M]. Stereochemistry of the 3'-OH had much less of an activity role than that of the 4'-OH, in contrast to existing models of "sugar-site" binding.

Cardiac glycosides: 2. Synthesis of glucose and galactose analogs.

Five cardioactive steroid genins 1a to 5a of widely varying C17 beta-side groups were converted by modified Koenigs-Knorr reactions into the corresponding beta-D-glucosides 1c to 5c and beta-D-galactosides 1e to 5e. The genins included digitoxigenin (3 beta, 14-dihydroxy-5 beta, 14 beta-card-20-(22)-enolide, 1a); (20R)-20, 22-dihydrodigitoxigenin (3 beta, 14-dihydroxy-5 beta, 14 beta, 20R-cardanolde, 2a); 3 beta, 14-dihydroxy-22-methylene-5 beta, 14 beta, 20S-cardanolide (3a); methyl 3 beta, 14-dihydroxy-5 beta, 14 beta-pregn-20(E)-ene-21-carboxylate (4a); and methyl 3 beta, 14-dihydroxy-21-methylene-5 beta, 14 beta-pregnane-21-carboxylate (5a).

Interaction of (Na+,K+)-ATPases and digitalis genins. A general model for inhibitory activity.

Previous models of digitalis genin interaction with the (Na+,K+)-ATPase system (the putative receptor for such drugs) were deficient in explaining the (Na+,K+)-ATPase inhibitory activity of a number of digitalis genin analogues. With rat brain (Na+,K+)-ATPase we observed that the C-17 side chain carbonyl (C = O) oxygen distance of a given genin in relation to its position in the reference compound digitoxigenin was the primary determinant of its biological activity. With a number of genin analogues, we observed a strict correlation of this structural parameter with its binding site compatibility as well as inhibitory potency with respect to the (Na+,K+)-ATPase. In every case the correlation to inhibition data was obtained using a minimum energy conformation for the genin structure. The general applicability of that model is now proposed based on the following observations. The carbonyl oxygen position versus the biological activity relationship fully holds with (Na+,K+)-ATPase preparations from other tissues and species and also when different binding conditions are used for the enzyme genin interaction. The relationship is equally valid for the K+-p-nitrophenyl phosphatase activity. Correlations of the data obtained under these various conditions provide further support for this relationship and for the concept that altered affinities of the enzyme for a given genin under different binding conditions reflect conformational variations of a single binding site.

Conformational analysis of sterols: comparison of X-ray crystallographic observations with data from other sources.

Cystallographic data on over 400 steroids collected in the Atlas of Steroid Structure provide information concerning preferred conformations, relative stabilities and substituent influence of the interactive potential of steroid hormones. Analysis of these data indicates that observed conformational details are intramolecularly controlled and that the influence of crystal packing forces is negligible. Crystallographic data on the orientation of the progesterone side chain contradict published force-field calculations. In 84 of 88 structures having a 20-one substituent, the C(16)-C(17)-C(20)-O(20) torsion angle is between 0 degrees and -46 degrees. The 4 torsion angles that lie outside this range do so because of a 16 beta-substituent and not because of crystal packing forces. Not one of the 88 structures is found to have a conformation in which the C(16)-C(17)-C(20)-O(20) torsion angle is within +/- 15 degrees of the most commonly calculated minimum energy value. The narrow range of side chain conformations seen in very different crystalline environments in the 88 crystal structure determinations and the predictable substituent influence apparent in the data strongly suggest that crystallographically observed conformers seldom deviate from minimum energy positions, regardless of hypothetical broad energy minima, metastable states and small barriers to rotation. The 96 crystallographically independent determinations of the cholestane 17-side chain show that the chain has 4 principal conformations (A:B:C:D), occurring in the ratio 69:8:8:11. Although the fully extended side chain is clearly the energetically most favored one, in 16 observations of cholesterol itself only 6 are in the extended conformation. Some of the correlated conformational changes in the chains can be rationalized on the basis of model studies, but others apparently result from subtle intramolecular forces. The unsaturated B ring provides another element of flexibility in the structure of cholesterol. The 5-ene B ring is normally observed in an 8 beta, 9 alpha-half-chair conformation. However, in structures containing more than one molecular in the crystallographic asymmetric unit, at least one of the 2 molecules is found to differ significantly from this form. It may be that this inherent flexibility is responsible for the presence of conformationally distnct molecules in the same crystal. The intermolecular interaction observed in the crystal structure of cholesterol and its fatty acid derivatives illustrate the type of interaction between the steroid ring system and hydrocarbon chains that can be expected in membrane bilayers.

Steroid structure and function VII. remarkable estrogenicity of 3-hydroxy-9 beta-estra-1,3,5(10)-triene-11,17-dione.

Remarkably high estrogenic activity was observed for 3-hydroxy-9 beta-estra-1,3,5(10)-triene-11,17-dione despite its unusual bent conformation. The 9 alpha epimer of this compound has markedly less activity despite the fact that its overall shape is nearly identical to that of estrone. The potency of these compounds in enhancing uterine weight in Fischer rats and reducing ovarian weight in parabiosed rats was compared with that of estrone, and the structures were unambiguously identified by X-ray crystallographic study. The results underscore the importance of the phenolic ring A to estrogenic activity, and suggest a tolerance of the putative estrogenic receptor to flexibility in overall molecular shape.

Conformational analysis of synthetic androgens. IV. 1,2-seco-a-bisnor-5 alpha-androstan-17 beta-ol acetate.

The crystal and molecular structure of 1,2-seco-A-bisnor-5 alpha-androstan-17beta-ol acetate has been determined to evaluated the conformational importance of the intact steroid nucleus. The resulting tricyclic compound retains nearly the same steric profile for the remainder of the molecule when compared to the structures of dihydrotestosterone derivatives with intact A-rings. This may help to explain why these types of molecules retain a significant level of androgenic activity.