Structure-activity studies of enzyme substrates using model interaction calculations

The hydrolysis rates in a series of substituted N-acetylaminoacid methylesters with acylase I depend apparently on the binding strengths of the side chains to the enzyme surface. Using a truncated model of a possible enzyme binding site we calculate the binding energies for a series of straight chain substituents. Our calculations reveal a very good correlation between the calculated energies and the hydrolysis rates, indicating the value of our receptor model. We feel this to be another example of the validity of our receptor mapping using model interaction energy calculations based on the monopoles-bond polarizabilities method.     

Structure-activity studies on odor molecules using molecular connectivity

Several classes of odorants have been examined using a newly developed structural analysis known as molecular connectivity. Within each class significant correlations have been found between odor similarities to a reference standard and connectivity terms. In each case some interpretation is possible as to certain structure-activity relationships imparting odor.     

Structure-activity studies on mammalian adenosine kinase

The structure-activity relationship for Chinese hamster adenosine kinase (AK) was examined by making systematic deletions from the N- and C-terminal ends. The first 16 a.a. residues from the N-terminal end, which likely form a random coil, can be deleted without any effect on AK activity or stability. The successive removal of the next 11 residues, which stabilize the first beta structure of the protein, leads to a progressive loss of AK activity from 100 to about 3%. The loss in activity is accompanied by increasing thermal instability and a slight increase in the K(m) for adenosine. All deletions beyond residue M28, which should cause disruption of the tertiary structure, are devoid of AK activity. The residues at the C-terminal end form a substructure involved in the stability of the "adenosine 2 binding site" and removal of any residues results in significant loss of activity. Successive removal of the first 10 residues from this end causes progressive decrease in AK activity to about the 2% level, accompanied by a five-fold increase in the K(m) for ATP, supporting the view that the adenosine 2 binding site located near the C-terminal end is the ATP binding site. All deletions beyond residue R348, which forms two salt bridges with the ATP binding site, are inactive. Site-directed replacement of an aspartic acid residue (D316), which is postulated to function in the transfer of phosphate from ATP to adenosine by either asparagine or glutamic acid, leads to complete loss of activity, supporting the proposed role of D316 as the catalytic base.     

Structure-activity studies on high affinity NOP-active hexapeptides.

Nociceptin/orphanin FQ (N/OFQ) is a 17 amino acid peptide that is the endogenous ligand for the G-protein coupled receptor ORL1 (NOP), a member of the opioid receptor family. Although it is clear that this receptor system is involved in a variety of physiologic functions, including analgesia, the precise actions of N/OFQ remain largely uncharacterized. One reason for this has been limited number of high-affinity ligands to NOP, and particularly the lack of availability of useful specific antagonists. Herein, we describe the pharmacologic activity of a series of modified amino acid containing modifications of the hexapeptide Ac-RYYRWR-NH2, with high affinity for NOP. These compounds were tested for binding affinity using [3H]N/OFQ binding to human NOP in CHO cells, and functional activity by measuring stimulation of [35S]GTPgammaS-binding in CHO cell membranes. These studies suggest that each Arg of the hexapeptide is required to maintain high-binding affinity. The peptide maintains high affinity if the Tyr2 or Tyr3 are modified, but at least one of these residues must maintain its hydroxyl group or there is a large decrease in intrinsic activity of the peptide.     

Structure-activity studies on cysteine-substituted neurokinin A analogs.

A complete series of analogs of tyrosine modified neurokinin A ([Tyr1]-NKA or [Tyr0]-NKA) has been synthesized by substituting each natural residue with 1-Cys. These analogs were tested for their ability to bind recombinant neurokinin-2 (NK-2) receptor. Substitution of Phe6 with Cys completely abolished binding of the analog to the receptor. Substitution of residues in the carboxyl-terminal region of the peptide (Met10, Leu9, Gly8, Val7) and Asp4 with Cys gave reductions in binding affinity of between 23- and 250-fold. Molecular dynamics simulations of these analogs suggest that changes in peptide structure and flexibility are not large contributors to the losses in receptor binding affinity. Reductions in binding affinity are therefore more confidently ascribed to losses of peptide-receptor interactions.     

Structure-activity studies of β-carboline analogs

A number of β-carboline analogs have been obtained or synthesized, and their $\text{in vitro}$ receptor affinities and $\text{in vivo}$ antagonist activities determined. The choice of analogs was made in order to explore the importance of the N₉-H, the aromatic nitrogen and the C₃-ester moiety for high-receptor affinity and antagonist activity of this class of benzodiazepine antagonist. Among the analogs investigated, we describe the properties of 3-cyano-β-carboline ($\text{lh}$), the first potent β-carboline antagonist without a carbonyl at the C₃-position.The results obtained indicate: (1) Specific interactions of the C₃-substituent with key cationic receptor sites rather than electron-withdrawing properties are important for high-receptor affinity and antagonist activity. (2) Specific in-plane interactions of the atomatic nitrogen with a cationic receptor site, rather than stacking with neutral aromatic residues of the receptor are also important for high affinity and antagonist activity. (3) While the presence of an N₉H enhances receptor affinity, interaction with an anionic receptor site does not appear essential for antagonist activity.     

Structure-activity relationship studies of flavopiridol analogues

Cyclin dependent kinases (CDKs) along with the complementary cyclins form key regulatory checkpoint controls on the cell cycle. Flavopiridol is a synthetic flavone that shows potent and selective cyclin-dependent kinase inhibitory activity. In this paper, we report modifications of the 3-hydroxy-1-methylpiperidinyl (D ring) of flavopiridol and their effect on CDK inhibitory activity.     

Structure-activity studies of RNA-binding oxazolidinone derivatives

The structure-activity relationship of a series of oxazolidinones binding to T-box riboswitch antiterminator RNA has been investigated. Oxazolidinones differentially substituted at C-5 were prepared and the ligand-induced fluorescence resonance energy transfer (FRET) changes in FRET-labeled antiterminator model RNA were assayed. Both qualitative and quantitative analysis of the structure-activity relationship indicate that hydrogen bonding and hydrophobic properties play a significant role in ligand binding.     

Power calculations for genetic association studies using estimated probability distributions

The determination of the power of-or of an appropriate sample size for-genetic association studies that exploit linkage disequilibrium requires many assumptions. Some of the more important assumptions include the linkage-disequilibrium strength among alleles at the observed marker-locus sites and a potential trait-influencing locus, the frequencies of the marker locus and trait-influencing alleles, and the ultimate density of the marker locus "map" (i.e., the number of bases between marker loci) necessary in order to identify, with some confidence, trait-influencing alleles. I consider an approach to assessment of the power and sample-size requirements of genetic case-control association study designs that makes use of empirically derived estimates of the distributions of important parameters often assumed to take on arbitrary values. My proposed methodology is extremely general and flexible and ultimately can provide realistic answers to questions such as "How many markers and/or how many individuals might it take to identify, with confidence, a disease gene, via linkage-disequilibrium and association methods from a candidate region or whole genome perspective?" I showcase aspects of the proposed methodology, using information abstracted from the literature.     

Structure-activity studies on adipokinetic hormones in Manduca sexta.

Structure-activity studies were performed for adipokinetic hormone (AKH) in Manduca sexta. Seven naturally occurring and four synthetic peptides of the red pigment concentrating hormone (RPCH)/AKH family were tested in larvae of M. sexta for activation of glycogen phosphorylase in fat body. pGlu at the N-terminal was found to be important for activity of peptides; however, Manduca AcGly1AKH is partially active. The amino acids at all positions appear to be of importance for activity, with the possible exception of the two serine residues in positions six and seven. Generally, the more amino acids are exchanged, the less the peptide will bind to the receptor. In M. sexta a beta-bend appears not to be important for the binding of peptides. Peptides ten amino acids long appear to be more active than shorter ones.     

Ligand recognition by chloroperoxidase using molecular interaction fields and quantum chemistry calculations

We recently reported that chloroperoxidase (CPO) from Caldariomyces fumago showed atypical kinetic behavior for the oxidation of 4,6 dimethyl dibenzothiophene (DMDBT). In this work, we undertake the theoretical study of DMDBT docking into CPO's active site, in order to clarify its binding capacity and affinity using molecular interaction fields and quantum mechanical procedure. The results revealed that CPO has two substrate binding sites with similar affinities for DMDBT. This finding suggests that the atypical kinetic behavior of CPO for the oxidation of DMDBT might be due to the simultaneous binding of two DMDBT molecules during its reaction cycle. Finally, we extend these results to carbazole, a nitrogen-containing PAH, through experimental and theoretical studies.     

In vitro studies and model calculations on the kinetics of fibrinolysis with streptokinase]

A method of measuring the distribution kinetics of radioactive 51Cr-EDTA between human plasma and a fibrinoclot are represented. In the presence of streptokinase the speed of distribution is a measure for the progressing of fibrinolysis. The results of measurement as well as the model calculations on the kinetics of fibrinolysis by streptokinase with physiologically relevant data are compared with those observations on the mechanism of thrombolysis obtained from biochemical findings. These results indicate that at the dissolution speeds of the clots observed clinically or experimentally the diffusion of streptokinase activator complexes into the clot seems to play no important role.     

Structure-activity studies of vasoactive intestinal polypeptide.

This report explores the potential side-chain functional groups required for interaction of the bronchodilator neuropeptide, vasoactive intestinal peptide (VIP), with its receptor. The binding affinity and biological activity of native VIP have been found to be sensitive to the removal of amino- and carboxyl-terminal residues. This data suggests that elements within the entire primary sequence of the VIP molecule appear to be necessary for recognition by VIP receptors. The introduction of alanine residues substituted into the VIP molecule is utilized to probe for side-chain functional groups that are crucial for eliciting high receptor binding affinity in vitro and high biological potency in vivo. The VIP pharmacophore appears to be identical in guinea pig lung and human lung and consists of multiple binding sites most likely involving positions Asp3, Phe6, Thr7, Tyr10, Tyr22, and Leu23. These findings could be exploited to enhance the biological potency of VIP by increasing the binding energy at these positions.     

Structure-activity studies with cholecystokinin on gastric secretion in the cat

The influence of the position of the sulphate group in CCK on its gastric acid and pepsin stimulating activities was investigated in conscious cats with gastric fistulae. In Boc-CCK7, substitution of tyrosine-SO₃H by ε-hydroxynorleucine-SO₃H, an aliphatic amino acid approximating the length of tyrosine, enhanced acid secretory potency, whilst similar substitution by serine-SO₃H reduced potency, possibly due to the serine residue holding the sulphate group closer to the peptide backbone. Desulphation of Ser-CCK6 reduced acid secretory potency indicating that the known loss of potency upon desulphation of CCK-like peptides is not wholly dependent upon the presence of tyrosine residue in position 7. Sulphated CCK-like peptides are partial agonists of pepsin secretion, and desulphation confers full agonist activity. Analogues of CCK with serine or ε-hydroxynorleucine substituting for tyrosine, whether sulphated or not, showed full agonist activity in stimulating pepsin secretion. These data suggest the presence of the aromatic tyrosine residue, as well as sulphation, to be a necessary prerequisite for pepsin partial agonist activity in CCK-like peptides.     

Interpretation of energy-transfer experiments by theoretical studies of model compounds using semiempirical potential functions. II. Monte Carlo calculations on oligopeptides

The properties relevant to nonradiative energy transfer have been computed in the unperturbed chain model for oligopeptides composed of from 4 to 21 residues of the formula Tyr-(Ala)_n-Tyr and Trp-(X)_n-Tyr, X being either Ala or Gly. A Monte Carlo method has been used for the generation of the chains. The relation between the distribution functions of the distances between the luminophores and the various properties in energy transfer has been examined for chains of different lengths and compositions. The averge of the orientation factor κ² has been computed as a functions of chain length both for molecules in a randomly coiled state and for molecules with backbones in a well-defined three-dimensional structure. The various averaging regimes of energy-transfer efficiency and of fluorescence decay are compared. Theoretical curves relating experimental efficiencies to the mean distance between the luminophores are proposed.     

Sample size calculations for designing clinical proteomic profiling studies using mass spectrometry

In cancer clinical proteomics, MALDI and SELDI profiling are used to search for biomarkers of potentially curable early-stage disease. A given number of samples must be analysed in order to detect clinically relevant differences between cancers and controls, with adequate statistical power. From clinical proteomic profiling studies, expression data for each peak (protein or peptide) from two or more clinically defined groups of subjects are typically available. Typically, both exposure and confounder information on each subject are also available, and usually the samples are not from randomized subjects. Moreover, the data is usually available in replicate. At the design stage, however, covariates are not typically available and are often ignored in sample size calculations. This leads to the use of insufficient numbers of samples and reduced power when there are imbalances in the numbers of subjects between different phenotypic groups. A method is proposed for accommodating information on covariates, data imbalances and design-characteristics, such as the technical replication and the observational nature of these studies, in sample size calculations. It assumes knowledge of a joint distribution for the protein expression values and the covariates. When discretized covariates are considered, the effect of the covariates enters the calculations as a function of the proportions of subjects with specific attributes. This makes it relatively straightforward (even when pilot data on subject covariates is unavailable) to specify and to adjust for the effect of the expected heterogeneities. The new method suggests certain experimental designs which lead to the use of a smaller number of samples when planning a study. Analysis of data from the proteomic profiling of colorectal cancer reveals that fewer samples are needed when a study is balanced than when it is unbalanced, and when the IMAC30 chip-type is used. The method is implemented in the clippda package and is available in R at: http://www.bioconductor.org/help/bioc-views/release/bioc/html/clippda.html.