Structure-activity Maps Research Articles

Non-enzymatic properties of Proteus mirabilis urease subunits

Ureases are moonlighting proteins displaying non-catalytic properties, including platelet activation, antifungal and entomotoxic effects. The structure-activity mapping of these properties is poorly developed. Proteus mirabilis urease (PMU) consists of three subunits, PmUreα, PmUreβ and PmUreγ, in an (αβγ)3 organization. In order to study the structure-activity relationships of PMU we obtained the recombinant subunits of this urease and evaluated their biological activities. The holo-urease promoted platelet aggregation, and toxicity in fungal and insect models. Similar to Jaburetox, a plant urease-derived polypeptide, PmUreβ showed the highest toxicity against yeasts and insects, and activated human platelets. PmUreγ and PmUreα presented insecticidal action upon injection. In addition, only PmUreγ and PmUreβ promote hemocytes aggregation. Bioinformatics analyses revealed gene/segment duplication and evolutionary divergence among ureases. Our findings show that PmUreβ (and probably its counterparts in other ureases) carries most of the non-enzymatic activities of these proteins.

Structure-activity mapping of ARHGAP36 reveals regulatory roles for its GAP homology and C-terminal domains.

ARHGAP36 is an atypical Rho GTPase-activating protein (GAP) family member that drives both spinal cord development and tumorigenesis, acting in part through an N-terminal motif that suppresses protein kinase A and activates Gli transcription factors. ARHGAP36 also contains isoform-specific N-terminal sequences, a central GAP-like module, and a unique C-terminal domain, and the functions of these regions remain unknown. Here we have mapped the ARHGAP36 structure-activity landscape using a deep sequencing-based mutagenesis screen and truncation mutant analyses. Using this approach, we have discovered several residues in the GAP homology domain that are essential for Gli activation and a role for the C-terminal domain in counteracting an N-terminal autoinhibitory motif that is present in certain ARHGAP36 isoforms. In addition, each of these sites modulates ARHGAP36 recruitment to the plasma membrane or primary cilium. Through comparative proteomics, we also have identified proteins that preferentially interact with active ARHGAP36, and we demonstrate that one binding partner, prolyl oligopeptidase-like protein, is a novel ARHGAP36 antagonist. Our work reveals multiple modes of ARHGAP36 regulation and establishes an experimental framework that can be applied towards other signaling proteins.

Nonproteinogenic deep mutational scanning of linear and cyclic peptides

High-resolution structure-activity analysis of polypeptides requires amino acid structures that are not present in the universal genetic code. Examination of peptide and protein interactions with this resolution has been limited by the need to individually synthesize and test peptides containing nonproteinogenic amino acids. We describe a method to scan entire peptide sequences with multiple nonproteinogenic amino acids and, in parallel, determine the thermodynamics of binding to a partner protein. By coupling genetic code reprogramming to deep mutational scanning, any number of amino acids can be exhaustively substituted into peptides, and single experiments can return all free energy changes of binding. We validate this approach by scanning two model protein-binding peptides with 21 diverse nonproteinogenic amino acids. Dense structure-activity maps were produced at the resolution of single aliphatic atom insertions and deletions. This permits rapid interrogation of interaction interfaces, as well as optimization of affinity, fine-tuning of physical properties, and systematic assessment of nonproteinogenic amino acids in binding and folding.

Subtle changes in endochin-like quinolone structure alter the site of inhibition within the cytochrome bc1 complex of Plasmodium falciparum.

The cytochrome bc1 complex (cyt bc1) is the third component of the mitochondrial electron transport chain and is the target of several potent antimalarial compounds, including the naphthoquinone atovaquone (ATV) and the 4(1H)-quinolone ELQ-300. Mechanistically, cyt bc1 facilitates the transfer of electrons from ubiquinol to cytochrome c and contains both oxidative (Qo) and reductive (Qi) catalytic sites that are amenable to small-molecule inhibition. Although many antimalarial compounds, including ATV, effectively target the Qo site, it has been challenging to design selective Qi site inhibitors with the ability to circumvent clinical ATV resistance, and little is known about how chemical structure contributes to site selectivity within cyt bc1. Here, we used the proposed Qi site inhibitor ELQ-300 to generate a drug-resistant Plasmodium falciparum clone containing an I22L mutation at the Qi region of cyt b. Using this D1 clone and the Y268S Qo mutant strain, P. falciparum Tm90-C2B, we created a structure-activity map of Qi versus Qo site selectivity for a series of endochin-like 4(1H)-quinolones (ELQs). We found that Qi site inhibition was associated with compounds containing 6-position halogens or aryl 3-position side chains, while Qo site inhibition was favored by 5,7-dihalogen groups or 7-position substituents. In addition to identifying ELQ-300 as a preferential Qi site inhibitor, our data suggest that the 4(1H)-quinolone scaffold is compatible with binding to either site of cyt bc1 and that minor chemical changes can influence Qo or Qi site inhibition by the ELQs.

A Quantitative Structure-Activity Relationship Model

Structure-activity relationships were studied for a series of 46 2.6-dimethyl-3.5-dicabomethoxy-4-phenyl-1.4-dihydropyridine derivatives by means of multiple linear regression (MLR) and artificial neural network (ANN) techniques. The values of log (1/EC50), which represents the 50% effective concentration for blocking the Ca2+ channel of the studied compounds were correlated with the descriptors encoding the chemical structures. Using the pertinent descriptors revealed by the regression analysis, a correlation coefficient of 0.99 (s = 0.23) for the training set (n = 46) was obtained for the ANN using the Levenberg-Marquardt algorithm with a 3-10-1 configuration. The results obtained from this study indicate that the activity of 2.6-dimethyl-3.5-dicabomethoxy-4-phenyl-1.4-dihydropyridine derivatives is strongly dependent on molar refractivity (MR), electronic factors (especially on the connectivity indices (IC0)) and hydrogen-bond donor's (HBD) of the molecule. Comparison of the descriptor's contribution obtained with MLR and ANN models indicates the presence of non-linearity in the data and the interaction effect between them since the efficiency of these descriptors was increased by the ANN model.On the other hand, we have used a new, robust structure-activity mapping technique, a Bayesian-regularized neural network, to develop a quantitative structure-activity relationship (QSAR) model, and the ability of the model was tested by using the cross validation technique. The results show that the method is robust and reliable and gives good results. Comparisons of Bayesian neural net models with those derived by classical MLR model analysis showed its superiority in generalization.

39 Structure-activity relationship studies of nucleoside analogs with anti-HIV activity using structure-activity maps : R.R. Parakulam, M.L. Lesniewski, and C.-c. Tsai Department of Chemistry, Kent State University, Kent, Ohio 44242 USA

39 Structure-activity relationship studies of nucleoside analogs with anti-HIV activity using structure-activity maps : R.R. Parakulam, M.L. Lesniewski, and C.-c. Tsai Department of Chemistry, Kent State University, Kent, Ohio 44242 USA

QSAR Studies of Antiviral Agents Using Molecular Similarity Analysis and Structure-Activity Maps

Quantitative structure-activity relationships (QSAR) were developed for nucleoside analogs with anti-HIV activity. These compounds were investigated to determine the correlation of structure and toxicity/activity using molecular similarity analysis and structure-activity maps. A multiple-formula approach was used to perform quantitative molecular similarity analysis (QMSA) and QSAR study. Molecular descriptors such as number of atoms and bonds of a molecule (NAB), maximum common substructure (MaCS), and molecular similarity index (MSI) were used in our structure-activity relationship study. The MaCS of two molecules is defined as the substructure with the greatest NAB value common to both molecules. The MSI of two molecules X and Y is defined as MSI (X,Y) = [MaCS (X,Y) /NAB (X) ] X [MaCS (X,Y) /NAB (Y) ]. MaCS and MSI quantify the similarity between two molecular structures. Structure-activity maps (structure-toxicity map and structure-antiviral map) and QMSA were used to determine the site and type of modification for reduced toxicity and improved activity of new compounds.

Spectrometrically monitored selection experiments: quantitative laser desorption mass spectrometry of small chemical libraries

Selection experiments involving chemical libraries are routinely used in the pharmaceutical industry for finding and optimizing lead compounds. In principle, almost any process involving a binding event or a reaction could be probed systematically with chemical libraries prepared by combinatorial synthesis. Traditionally, however, the vast majority of library members cannot be monitored during the selection, making a systematic correlation of structure and activity difficult. To interpret selection experiments on the level of all library components, monitoring technologies are required that give a unique and quantitative spectroscopic signal for every compound in a mixture. Quantitative matrix-assisted laser desorption mass spectrometry of libraries of porphyrins and peptide-DNA hybrids consisting of 2-35 compounds is described. Porphyrin libraries were subjected to in vitro selections for liposome incorporation and binding to a protein pocket. It was shown that mesohydroxyphenyl substituted porphyrins, known high activity photosensitizers of tumors, are preferentially incorporated in liposome membranes. A mixture of peptide-DNA hybrids was assayed for the nuclease stability of its components. Small libraries of non-isobaric compounds can be exhaustively or near-exhaustively monitored by mass spectrometry. Monitored selection experiments can yield detailed structure-activity maps in a single experiment, speeding up drug discovery and the probing of biochemically relevant recognition events. It is proposed that monitored assays for target binding, membrane partitioning, and biostability could be run in parallel, to select drug candidates combining several favorable properties in 'multidimensional' selection experiments.

QSAR study of nucleoside analogs with anti-HIV activity using molecular similarity analysis and structure-activity maps

QSAR study of nucleoside analogs with anti-HIV activity using molecular similarity analysis and structure-activity maps

Structure-activity maps for visualizing the graph variables arising in drug design.

Structure-activity problems are characterized by the topological and topographical character of the structural information determining the activity. Traditional statistical methodology requires that this predictive information be mapped to a vector space. To circumvent this vexing conversion of structural information to vector form, the edge-deletion metric is defined on the space of chemical graphs that defines the topology of the molecules. This paper proposes structure-activity maps and transformation-effect maps for directly visualizing the structure-activity relationships. The maps are illustrated using the hypotensive activities of clonidine analogs and the sweet taste of Perillartine analogs.

Visualizing relative occurrences in metabolic transformations of xenobiotics using structure-activity maps.

Structure maps are presented as an efficient means of indicating structure-reactivity relationships in metabolic pathway databases. The relative occurrence of N-demethylation and N-oxidation of N-methyl tertiary amines was examined using the structure map methodology. A new family of reaction site representations, the n-level representations, was developed to describe the N-methyl reaction sites of the compounds in the data set. It was possible to differentiate N-demethylation and N-oxidation reaction sites using a structure map constructed from a 3-level representation of the reaction sites.

Structure-function mapping of interleukin 1 precursors. Cleavage leads to a conformational change in the mature protein.

The two interleukin 1 (IL-1) genes (IL-1 alpha and beta) encode 31-kDa precursor molecules, which are cleaved upon secretion to generate the mature, active, carboxyl-terminal 17-kDa proteins. The IL-1 beta precursor is inactive, whereas the IL-1 alpha precursor is as active as the mature IL-1 alpha. In this report, we demonstrate that when either of the recombinant precursors is processed to the mature form, the mature region undergoes a conformational change from a proteinase K-sensitive structure to one that is proteinase K-insensitive. In addition, cysteine residues that are exposed to solvent in the IL-1 beta precursor become buried in the mature protein. Limited structure-activity mapping of the IL-1 beta precursor indicates that the amino-terminal 76 residues are responsible for the conformational change, whereas the most dramatic change in biological activity occurs after further removal of residues 77-94. These findings suggest that the altered structure of the mature region in precursor IL-1s has been conserved for some function. Denaturation/renaturation experiments implicate the precursor domain in protein folding, and by analogy with signal-directed secretory proteins, the unique conformation of the precursors may play a role in IL-1 secretion.