Identifying protein targets of bioactive small molecules often requires complex, lengthy development of affinity probes. We present a method for stochastic modification of small molecules of interest with a photoactivatable phenyldiazirine linker. The resulting isomeric mixture is conjugated to a hydrophilic copolymer decorated with biotin and a fluorophore. We validated this approach using known inhibitors of several medicinally relevant enzymes. At least a portion of the stochastic derivatives retained their binding to the target, enabling target visualization, isolation, and identification. Moreover, the mix of stochastic probes could be separated into fractions and tested for binding affinity. The structure of the active probe could be determined and the probe resynthesized to improve binding efficiency. Our approach can thus enable rapid target isolation, identification, and visualization, while providing information required for subsequent synthesis of an optimized probe.
- MeSH
- afinitní značky chemická syntéza chemie účinky záření MeSH
- aspartátové endopeptidasy antagonisté a inhibitory chemie MeSH
- biotin chemie MeSH
- diazomethan analogy a deriváty chemická syntéza účinky záření MeSH
- fluoresceiny chemie MeSH
- fluorescenční barviva chemie MeSH
- glutamátkarboxypeptidasa II antagonisté a inhibitory chemie MeSH
- hmotnostní spektrometrie metody MeSH
- inhibitory enzymů chemická syntéza chemie účinky záření MeSH
- konfokální mikroskopie metody MeSH
- kyseliny polymethakrylové chemie MeSH
- lidé MeSH
- membránové proteiny antagonisté a inhibitory chemie MeSH
- nádorové buněčné linie MeSH
- proteomika metody MeSH
- serinové endopeptidasy chemie MeSH
- ultrafialové záření MeSH
- želatinasy antagonisté a inhibitory chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Nepenthesins are aspartic proteases secreted by carnivorous pitcher plants of the genus Nepenthes. They significantly differ in sequence from other plant aspartic proteases. This difference, which provides more cysteine residues in the structure of nepenthesins, may contribute to their unique stability profile. Recombinantly produced nepenthesin 1 (rNep1) from N. gracilis in complex with pepstatin A was crystallized under two different crystallization conditions using a newly formulated low-pH crystallization screen. The diffraction data were processed to 2.9 and 2.8 Å resolution, respectively. The crystals belonged to space group P212121, with unit-cell parameters a = 86.63, b = 95.90, c = 105.40 Å, α = β = γ = 90° and a = 86.28, b = 97.22, c = 103.78 Å, α = β = γ = 90°, respectively. Matthews coefficient and solvent-content calculations suggest the presence of two molecules of rNep1 in the asymmetric unit. Here, the details of the crystallization experiment and analysis of the X-ray data are reported.
Secreted aspartic proteases (Saps) are extracellular proteolytic enzymes that enhance the virulence of Candida pathogens. These enzymes therefore represent possible targets for therapeutic drug design. Saps are inhibited by nanomolar concentrations of the classical inhibitor of aspartic proteases pepstatin A and also by the inhibitors of the HIV protease, but with the K(i) of micromolar values or higher. To contribute to the discussion regarding whether HIV protease inhibitors can act against opportunistic mycoses by the inhibition of Saps, we determined the structure of Sapp1p from Candida parapsilosis in complex with ritonavir (RTV), a clinically used inhibitor of the HIV protease. The crystal structure refined at resolution 2.4 Å proved binding of RTV into the active site of Sapp1p and provided the structural information necessary to evaluate the stability and specificity of the protein-inhibitor interaction.
- MeSH
- aspartátové endopeptidasy antagonisté a inhibitory chemie MeSH
- Candida enzymologie MeSH
- fungální proteiny antagonisté a inhibitory chemie MeSH
- inhibitory HIV-proteasy chemie farmakologie MeSH
- krystalografie rentgenová MeSH
- molekulární modely MeSH
- ritonavir chemie farmakologie MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Opportunistic pathogens of the genus Candida cause infections representing a major threat to long-term survival of immunocompromised patients. Virulence of the Candida pathogens is enhanced by production of extracellular proteolytic enzymes and secreted aspartic proteases (Saps) are therefore studied as potential virulence factors and possible targets for therapeutic drug design. Candida parapsilosis is less invasive than C. albicans, however, it is one of the leading causative agents of yeast infections. We report three-dimensional crystal structure of Sapp1p from C. parapsilosis in complex with pepstatin A, the classical inhibitor of aspartic proteases. The structure of Sapp1p was determined from protein isolated from its natural source and represents the first structure of Sap from C. parapsilosis. Overall fold and topology of Sapp1p is very similar to the archetypic fold of monomeric aspartic protease family and known structures of Sap isoenzymes from C. albicans and Sapt1p from C. tropicalis. Structural comparison revealed noticeable differences in the structure of loops surrounding the active site. This resulted in differential character, shape, and size of the substrate binding site explaining divergent substrate specificities and inhibitor affinities. Determination of structures of Sap isoenzymes from various species might contribute to the development of new Sap-specific inhibitors.
- MeSH
- aspartátové endopeptidasy chemie MeSH
- Candida enzymologie patogenita MeSH
- fungální proteiny chemie MeSH
- katalytická doména MeSH
- konformace proteinů MeSH
- krystalografie rentgenová MeSH
- pepstatiny chemie MeSH
- sbalování proteinů MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- Publikační typ
- práce podpořená grantem MeSH
Secreted aspartic proteases (Saps) of pathogenic Candida spp. represent a specific target for antifungal drug development. We synthesized a series of peptidomimetic inhibitors with different isosteric groups and modifications at individual positions and tested them with purified Saps from C. albicans (Sap2p), C. tropicalis (Sapt1p), and C. parapsilosis (Sapp1p). The kinetic parameters indicated that all three proteases prefer binding of inhibitors containing bulky hydrophobic residues between positions P3 and P3'. The most divergent specificity was found for Sapp1p. The sequence alignment of Sap2p, Sapt1p, and Sapp1p, and homology modeling of Sapp1p with the crystal structure of Sapt1p and the complex of Sap2p with a peptidomimetic inhibitor showed that the overall folds of Sap2p, Sapt1p, and Sapp1p are similar. However, the N- and C-terminal loops formed by disulfide bonds between residues 47-53 and 258-292 are significantly shorter in Sapp1p, and a unique insertion following Tyr 129 in Sapp1p results in the formation of a loop that can interact with inhibitor residues. These Sapp1p structural differences might lead to its altered susceptibility to inhibition.
- MeSH
- aspartátové endopeptidasy chemie účinky léků MeSH
- Candida enzymologie MeSH
- financování organizované MeSH
- inhibitory proteas farmakologie chemická syntéza chemie MeSH
- izoenzymy chemie účinky léků MeSH
- molekulární konformace MeSH
- molekulární modely MeSH
- molekulární sekvence - údaje MeSH
- sekvence aminokyselin MeSH
- sekvenční homologie aminokyselin MeSH
- stereoizomerie MeSH
- substrátová specifita MeSH
- terciární struktura proteinů MeSH
- vztahy mezi strukturou a aktivitou MeSH
HIV protease (PR) represents a prime target for rational drug design, and protease inhibitors (PI) are powerful antiviral drugs. Most of the current PIs are pseudopeptide compounds with limited bioavailability and stability, and their use is compromised by high costs, side effects, and development of resistant strains. In our search for novel PI structures, we have identified a group of inorganic compounds, icosahedral metallacarboranes, as candidates for a novel class of nonpeptidic PIs. Here, we report the potent, specific, and selective competitive inhibition of HIV PR by substituted metallacarboranes. The most active compound, sodium hydrogen butylimino bis-8,8-[5-(3-oxa-pentoxy)-3-cobalt bis(1,2-dicarbollide)]di-ate, exhibited a K(i) value of 2.2 nM and a submicromolar EC(50) in antiviral tests, showed no toxicity in tissue culture, weakly inhibited human cathepsin D and pepsin, and was inactive against trypsin, papain, and amylase. The structure of the parent cobalt bis(1,2-dicarbollide) in complex with HIV PR was determined at 2.15 A resolution by protein crystallography and represents the first carborane-protein complex structure determined. It shows the following mode of PR inhibition: two molecules of the parent compound bind to the hydrophobic pockets in the flap-proximal region of the S3 and S3' subsites of PR. We suggest, therefore, that these compounds block flap closure in addition to filling the corresponding binding pockets as conventional PIs. This type of binding and inhibition, chemical and biological stability, low toxicity, and the possibility to introduce various modifications make boron clusters attractive pharmacophores for potent and specific enzyme inhibition.
- MeSH
- aspartátové endopeptidasy chemie MeSH
- borany farmakologie chemická syntéza chemie MeSH
- financování organizované MeSH
- HIV-proteasa chemie MeSH
- inhibitory HIV-proteasy farmakologie chemická syntéza chemie MeSH
- krystalografie rentgenová MeSH
- kvantitativní vztahy mezi strukturou a aktivitou MeSH
- racionální návrh léčiv MeSH
- MeSH
- aspartátové endopeptidasy genetika chemie imunologie MeSH
- finanční podpora výzkumu jako téma MeSH
- HIV protilátky chemie imunologie MeSH
- HIV-proteasa imunologie MeSH
- lidé MeSH
- monoklonální protilátky chemie MeSH
- peptidy chemie imunologie MeSH
- sekvence aminokyselin MeSH
- zkřížené reakce MeSH
- Check Tag
- lidé MeSH
- MeSH
- aspartátové endopeptidasy farmakologie chemie MeSH
- cysteinové endopeptidasy farmakologie chemie MeSH
- endopeptidasy farmakologie chemie MeSH
- enzymy chemie klasifikace MeSH
- metaloendopeptidasy farmakologie chemie MeSH
- proteiny metabolismus MeSH
- serinové endopeptidasy farmakologie chemie MeSH
- Publikační typ
- přehledy MeSH
