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- Erratum: Design of mutation-resistant HIV protease inhibitors with the substrate envelope hypothesis (Chemical Biology and Drug Design (2007) 69, (298-313)Publication . Chellappan, Sripriya; Kairys, Visvaldas; Fernandes, Miguel X.; Schiffer, Celia; Gilson, Michael K.There is a clinical need for HIV protease inhibitors that can evade resistance mutations. One possible approach to designing such inhibitors relies upon the crystallographic observation that the sub strates of HIV protease occupy a rather constant region within the binding site. In particular, it has been hypothesized that inhibitors which lie within this region will tend to resist clinically relevant mutations. The present study offers the first pros pective evaluation of this hypothesis, via compu tational design of inhibitors predicted to conform to the substrate envelope, followed by synthesis and evaluation against wild-type and mutant pro teases, as well as structural studies of complexes of the designed inhibitors with HIV protease. The results support the utility of the substrate envel ope hypothesis as a guide to the design of robust protease inhibitors.
- HIV-1 protease inhibitors from inverse design in the substrate envelope exhibit subnanomolar binding to drug-resistant variantsPublication . Altman, Michael D.; Ali, Akbar; Kumar Reddy, G. S. Kiran; Nalam, Madhavi N. L.; Anjum, Saima Ghafoor; Cao, Hong; Chellappan, Sripriya; Kairys, Visvaldas; Fernandes, Miguel X.; Gilson, Michael K.; Schiffer, Celia A.; Rana, Tariq M.; Tidor, BruceThe acquisition of drug-resistant mutations by infectious pathogens remains a pressing health concern, and the development of strategies to combat this threat is a priority. Here we have applied a general strategy, inverse design using the substrate envelope, to develop inhibitors of HIV-1 protease. Structure-based computation was used to design inhibitors predicted to stay within a consensus substrate volume in the binding site. Two rounds of design, synthesis, experimental testing, and structural analysis were carried out, resulting in a total of 51 compounds. Improvements in design methodology led to a roughly 1000-fold affinity enhancement to a wild-type protease for the best binders, from a Ki of 30–50 nM in round one to below 100 pM in round two. Crystal structures of a subset of complexes revealed a binding mode similar to each design that respected the substrate envelope in nearly all cases. All four best binders from round one exhibited broad specificity against a clinically relevant panel of drug-resistant HIV-1 protease variants, losing no more than 6–13-fold affinity relative to wild type. Testing a subset of second-round compounds against the panel of resistant variants revealed three classes of inhibitors: robust binders (maximum affinity loss of 14–16-fold), moderate binders (35–80-fold), and susceptible binders (greater than 100-fold). Although for especially high-affinity inhibitors additional factors may also be important, overall, these results suggest that designing inhibitors using the substrate envelope may be a useful strategy in the development of therapeutics with low susceptibility to resistance.
- Using protein homology models for structure-based studies: approaches to model refinementPublication . Kairys, V.; Gilson, M. K.; Fernandes, Miguel XavierHomology modeling is a computational methodology to assign a 3-D structure to a target protein when experimental data are not available. The methodology uses another protein with a known structure that shares some sequence identity with the target as a template. The crudest approach is to thread the target protein backbone atoms over the backbone atoms of the template protein, but necessary refinement methods are needed to produce realistic models. In this mini-review anchored within the scope of drug design, we show the validity of using homology models of proteins in the discovery of binders for potential therapeutic targets. We also report several different approaches to homology model refinement, going from very simple to the most elaborate. Results show that refinement approaches are system dependent and that more elaborate methodologies do not always correlate with better performances from built homology models.
- Toward the design of mutation‐resistant enzyme inhibitors: further evaluation of the substrate envelope hypothesisPublication . Kairys, Visvaldas; Gilson, Michael K.; Lather, Viney; Schiffer, Celia A.; Fernandes, Miguel X.Previous studies have shown the usefulness of the substrate envelope concept in the analysis and prediction of drug resistance profiles for human immunodeficiency virus protease mutants. This study tests its applicability to several other thera peutic targets: Abl kinase, chitinase, thymidylate synthase, dihydrofolate reductase, and neuramini dase. For the targets where many (‡6) mutation data are available to compute the average mutation sen sitivity of inhibitors, the total volume of an inhibitor molecule that projects outside the substrate enve lope Vout, is found to correlate with average muta tion sensitivity. Analysis of a locally computed volume suggests that the same correlation would hold for the other targets, if more extensive muta tion data sets were available. It is concluded that the substrate envelope concept offers a promising and easily implemented computational tool for the design of drugs that will tend to resist muta tions. Software implementing these calculations is provided with the ’Supporting Information’.
- Insight into the role of N,N-dimethylaminoethyl methacrylate (DMAEMA) conjugation onto poly(ethylenimine): cell viability and gene transfection studiesPublication . Nouri, Alireza; Castro, Rita; Kairys, Visvaldas; Santos, José L.; Rodrigues, João; Li, Yulin; Tomás, HelenaIn the present study, the effect of N,N-dimethylaminoethyl methacrylate (DMAEMA) conjugation onto branched poly(ethylenimine) (PEI) with different grafting degree was examined for gene delivery applications. The DMAEMA-grafted-PEI conjugates were characterized and complexed with plasmid DNA (pDNA) at various concentrations, and the physicochemical properties, cell viability, and in vitro transfection efficiency of the complexes were evaluated in HEK 293T cells. Computational techniques were used to analyze the interaction energies and possible binding modes between DNA and conjugates at different grafting degrees. The cytotoxicity analysis and in vitro transfection efficiency of the conjugate/pDNA complexes exhibited a beneficial effect of DMAEMA conjugation when compared to PEI alone. The computational results revealed that the DNA/vector interaction energy decreases with increasing grafting degree, which can be associated to an enhanced release of the pDNA from the carrier once inside cells. The results indicate the significance of DMAEMA conjugation onto PEI as a promising approach for gene delivery applications.
- Design of mutation-resistant HIV protease inhibitors with the substrate envelope hypothesisPublication . Chellappan, Sripriya; Kiran Kumar Reddy, G. S.; Ali, Akbar; Nalam, Madhavi N. L.; Anjum, Saima Ghafoor; Cao, Hong; Kairys, Visvaldas; Fernandes, Miguel X.; Altman, Michael D.; Tidor, Bruce; Rana, Tariq M.; Schiffer, Celia A.; Gilson, Michael K.There is a clinical need for HIV protease inhibitors that can evade resistance mutations. One possible approach to designing such inhibitors relies upon the crystallographic observation that the sub strates of HIV protease occupy a rather constant region within the binding site. In particular, it has been hypothesized that inhibitors which lie within this region will tend to resist clinically relevant mutations. The present study offers the first pros pective evaluation of this hypothesis, via compu tational design of inhibitors predicted to conform to the substrate envelope, followed by synthesis and evaluation against wild-type and mutant pro teases, as well as structural studies of complexes of the designed inhibitors with HIV protease. The results support the utility of the substrate envel ope hypothesis as a guide to the design of robust protease inhibitors.
- Polyester Dendrimers Based on Bis-MPA for Doxorubicin DeliveryPublication . Gonçalves, Mara; Kairys, Visvaldas; Rodrigues, João; Tomás, HelenaAlthough doxorubicin (DOX) is one of the most used chemotherapeutic drugs due to its efficacy against a wide group of cancer types, it presents severe side effects. As such, intensive research is being carried out to find new nanoscale systems that can help to overcome this problem. Polyester dendrimers based on the monomer 2,2-bis- (hydroxymethyl)propionic acid (bis-MPA) are very promising systems for biomedical applications due to their biodegradability properties. In this study, bis-MPA-based dendrimers were, for the first time, evaluated as DOX delivery vehicles. Generations 4 and 5 of bis-MPA-based dendrimers with hydroxyl groups at the surface were used (B-G4-OH and B-G5-OH), together with dendrimers partially functionalized with amine groups (B-G4-NH2/OH and B-G5-NH2/OH). Partial functionalization was chosen because the main purpose was to compare the effect of different functional groups on dendrimers’ drug delivery behavior without compromising cell viability, which is often affected by dendrimers’ cationic charge. Results revealed that bis-MPA-based dendrimers were cytocompatible, independently of the chemical groups that were present at their surface. The B-G4-NH2/OH and B-G5-NH2/OH dendrimers were able to retain a higher number of DOX molecules, but the in vitro release of the drug was faster. On the contrary, the hydroxyl-terminated dendrimers exhibited a lower loading capacity but were able to deliver the drug in a more sustained manner. These results were in accordance with the cytotoxicity studies performed in several models of cancer cell lines and human mesenchymal stem cells. Overall, the results confirmed that it is possible to tune the drug delivery properties of bis-MPA-based dendrimers by modifying surface functionalization. Moreover, molecular modeling studies provided insights into the nature of the interactions established between the drug and the bis-MPA based dendrimersDOX molecules attach to their surface rather than being physically encapsulated.
- SitCon: binding site residue conservation visualization and protein sequence-to-function toolPublication . Kairys, Visvaldas; Fernandes, Miguel X.We introduce SitCon (SITe CONservation), a program designed to explore conservation of functionally important sites in a series of hypothetically homologous candidate protein structures, given amino acid sequence as an input. This can especially be useful when looking for an unknown function of a protein. SitCon exploits the fact that binding sites of proteins are preserved better than the overall residue sequence conservation. To test the capability of unknown function prediction, we randomly chose known function proteins from Caenorhabditis elegans genome. To imitate a behavior of an unknown function target, only the low homology proteins with 0.01 E-score 100 were analyzed as templates. Out of 29 enzyme targets, SitCon was able to provide various hints about their function in at least 69% of the cases. For the eight nonenzyme targets, the predictions matched in only 25% of the cases. SitCon was also tested for a capability to predict presence or absence of metal-containing heterogroups in the target enzymes with 80% success rate. Because this algorithm is not based on specific protein signatures, it may allow detection of overlooked relationships between proteins. SitCon is also very effective as a tool allowing visual comparison of binding site residue conservation between the target and homologous templates side-by-side.
- Enantioseparation and chiral recognition mechanism of new chiral derivatives of xanthones on macrocyclic antibiotic stationary phasesPublication . Fernandes, Carla; Tiritan, Maria Elizabeth; Cass, Quezia; Kairys, Visvaldas; Fernandes, Miguel Xavier; Pinto, MadalenaA chiral HPLC method using four macrocyclic antibiotic chiral stationary phases (CSPs) has been inves tigated for determination of the enantiomeric purity of fourteen new chiral derivatives of xanthones (CDXs). The separations were performed with the CSPs Chirobiotic T, Chirobiotic TAG, Chirobiotic V and Chirobiotic R under multimodal elution conditions (normal-phase, reversed-phase and polar ionic mode). The analyses were performed at room temperature in isocratic mode and UV and CD detection at a wavelength of 254 nm. The best enantioselectivity and resolution were achieved on Chirobiotic R and Chirobiotic T CSPs, under normal elution conditions, with RS ranging from 1.25 to 2.50 and from 0.78 to 2.06, respectively. The optimized chromatographic conditions allowed the determination of the enan tiomeric ratio of eight CDXs, always higher than 99%. In order to better understand the chromatographic behavior at a molecular level, and the structural features associated with the chiral recognition mech anism, computational studies by molecular docking were carried out using VDock. These studies shed light on the mechanisms involved in the enantioseparation for this important class of chiral compounds.
- Quantitative structure-activity relationship models with receptor-dependent descriptors for predicting peroxisome proliferator-activated receptor activities of thiazolidinedione and oxazolidinedione derivativesPublication . Lather, Viney; Kairys, Visvaldas; Fernandes, Miguel X.A quantitative structure–activity relationship study has been carried out, in which the relationship between the peroxisome proliferator-activated receptor a and the peroxisome proliferator activated receptor c agonistic activities of thiazo lidinedione and oxazolidinedione derivatives and quantitative descriptors, Vsite calculated in a receptor-dependent manner is modeled. These descriptors quantify the volume occupied by the optimized ligands in regions that are either com mon or specific to the superimposed binding sites of the targets under consideration. The quantita tive structure–activity relationship models were built by forward stepwise linear regression model ing for a training set of 27 compounds and vali dated for a test set of seven compounds, resulting in a squared correlation coefficient value of 0.90 for peroxisome proliferator-activated receptor a and of 0.89 for peroxisome proliferator-activated receptor c. The leave-one-out cross-validation and test set predictability squared correlation coeffi cient values for these models were 0.85 and 0.62 for peroxisome proliferator-activated receptor a and 0.89 and 0.50 for peroxisome proliferator-acti vated receptor c respectively. A dual peroxisome proliferator-activated receptor model has also been developed, and it indicates the structural features required for the design of ligands with dual peroxisome proliferator-activated receptor activity. These quantitative structure–activity relationship models show the importance of the descriptors here introduced in the prediction and interpretation of the compounds affinity and selectivity.