| 1. |
- Abdurakhmanov, Eldar, 1977-, et al.
(författare)
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Biophysical Mode-of-Action and Selectivity Analysis of Allosteric Inhibitors of Hepatitis C Virus (HCV) Polymerase
- 2017
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Ingår i: Viruses. - : MDPI AG. - 1999-4915. ; 9:6
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Tidskriftsartikel (refereegranskat)abstract
- Allosteric inhibitors of hepatitis C virus (HCV) non-structural protein 5B (NS5B) polymerase are effective for treatment of genotype 1, although their mode of action and potential to inhibit other isolates and genotypes are not well established. We have used biophysical techniques and a novel biosensor-based real-time polymerase assay to investigate the mode-of-action and selectivity of four inhibitors against enzyme from genotypes 1b (BK and Con1) and 3a. Two thumb inhibitors (lomibuvir and filibuvir) interacted with all three NS5B variants, although the affinities for the 3a enzyme were low. Of the two tested palm inhibitors (dasabuvir and nesbuvir), only dasabuvir interacted with the 1b variant, and nesbuvir interacted with NS5B 3a. Lomibuvir, filibuvir and dasabuvir stabilized the structure of the two 1b variants, but not the 3a enzyme. The thumb compounds interfered with the interaction between the enzyme and RNA and blocked the transition from initiation to elongation. The two allosteric inhibitor types have different inhibition mechanisms. Sequence and structure analysis revealed differences in the binding sites for 1b and 3a variants, explaining the poor effect against genotype 3a NS5B. The indirect mode-of-action needs to be considered when designing allosteric compounds. The current approach provides an efficient strategy for identifying and optimizing allosteric inhibitors targeting HCV genotype 3a.
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| 2. |
- Anscombe, Elizabeth, et al.
(författare)
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Identification and Characterization of an Irreversible Inhibitor of CDK2
- 2015
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Ingår i: Chemistry and Biology. - : Elsevier BV. - 1074-5521 .- 1879-1301. ; 22:9, s. 1159-1164
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Tidskriftsartikel (refereegranskat)abstract
- Irreversible inhibitors that modify cysteine or lysine residues within a protein kinase ATP binding site offer, through their distinctive mode of action, an alternative to ATP-competitive agents. 4-((6-(Cyclo-hexylmethoxy)-9H-purin-2-yl) amino) benzenesulfonamide (NU6102) is a potent and selective ATP-competitive inhibitor of CDK2 in which the sulfonamide moiety is positioned close to a pair of lysine residues. Guided by the CDK2/NU6102 structure, we designed 6-(cyclohexylmethoxy)-N-(4-(vinylsulfonyl) phenyl)-9H- purin-2-amine (NU6300), which binds covalently to CDK2 as shown by a co-complex crystal structure. Acute incubation with NU6300 produced a durable inhibition of Rb phosphorylation in SKUT-1B cells, consistent with it acting as an irreversible CDK2 inhibitor. NU6300 is the first covalent CDK2 inhibitor to be described, and illustrates the potential of vinyl sulfones for the design of more potent and selective compounds.
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| 3. |
- Belfrage, Anna Karin, et al.
(författare)
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Discovery of pyrazinone based compounds that potently inhibit the drug resistant enzyme variant R155K of the hepatitis C virus NS3 protease
- 2016
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Ingår i: Bioorganic & Medicinal Chemistry. - : Elsevier BV. - 0968-0896 .- 1464-3391. ; 24:12, s. 2603-2620
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Tidskriftsartikel (refereegranskat)abstract
- Herein, we present the design and synthesis of 2(1H)-pyrazinone based HCV NS3 protease inhibitors with variations in the C-terminus. Biochemical evaluation was performed using genotype 1a, both the wildtype and the drug resistant enzyme variant, R155K. Surprisingly, compounds without an acidic sulfonamide retained good inhibition, challenging our previous molecular docking model. Moreover, selected compounds in this series showed nanomolar potency against R155K NS3 protease; which generally confer resistance to all HCV NS3 protease inhibitors approved or in clinical trials. These results further strengthen the potential of this novel substance class, being very different to the approved drugs and clinical candidates, in the development of inhibitors less sensitive to drug resistance.
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| 4. |
- Belfrage, Anna Karin, 1977-, et al.
(författare)
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Pan-NS3 protease inhibitors of hepatitis C virus based on an R3-elongated pyrazinone scaffold
- 2018
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Ingår i: European Journal of Medicinal Chemistry. - : Elsevier. - 0223-5234 .- 1768-3254. ; 148, s. 453-464
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Tidskriftsartikel (refereegranskat)abstract
- Herein, we present the design and synthesis of 2(1H)-pyrazinone based HCV NS3 protease inhibitors and show that elongated R-3 urea substituents were associated with increased inhibitory potencies over several NS3 protein variants. The inhibitors are believed to rely on beta-sheet mimicking hydrogen bonds which are similar over different genotypes and current drug resistant variants and correspond to the beta-sheet interactions of the natural peptide substrate. Inhibitor 36, for example, with a urea substituent including a cyclic imide showed balanced nanomolar inhibitory potencies against genotype la, both wild-type (K-i=30 nM) and R155K (K-i=2 nM), and genotype 3a (K-i=5 nM).
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| 5. |
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| 6. |
- Danielson, U. Helena
(författare)
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Molecular Interaction Analysis for Discovery of Drugs Targeting Enzymes and for Resolving Biological Function
- 2015
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Ingår i: Multifaceted Roles Of Crystallography In Modern Drug Discovery. - Dordrecht : Springer Netherlands. - 9789401797245 - 9789401797191 - 9789401797184 ; , s. 223-240
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Konferensbidrag (refereegranskat)abstract
- Analysis of molecular interactions using surface plasmon resonance (SPR) biosensor technology has become a powerful tool for discovery of drugs targeting enzymes and resolving biological function. A major advantage of this technology over other methods for interaction analysis is that it can provide the kinetic details of interactions. This is a consequence of the time resolution of the analysis, which allows individual kinetic rate constants as well as affinities to be determined. A less commonly recognized feature of this technology is that it can reveal the characteristics of more complex mechanisms, e.g. involving multiple steps or conformations of the target or ligand, as well as the energetics, thermodynamics and forces involved.
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| 7. |
- Encarnação, João Crispim, Master, 1990-
(författare)
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Towards time-resolved molecular interaction assays in living bacteria
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Rare and neglected diseases such as multidrug resistant (MDR) tuberculosis, malaria and trypanosomiasis are re-emerging in Europe. New strategies are needed to accelerate drug discovery to fight these pathogens. AEGIS is a Pan-European project that combines different technologies to accelerate the discovery of molecules suitable for drug development in selected neglected diseases. This thesis is part of the AEGIS research area that considers time in a multidisciplinary approach, combining biology, physics and mathematics to provide tools to characterize biological events for improving drug development and information about the target diseases and lead compounds.Real-time cell binding assays (RT-CBA) of receptor-ligand interactions are fundamental in basic research and drug discovery. However, this kind of assays are still rare on living cells, especially in the microbiology field. In this project, we apply the same high-precision assay type on bacterial systems and explored the interior of the cell with a time resolved assay.The effect of temperature was evaluated in the RT-CBA using LigandTracer to ensure that it was possible to use the technology in a range of temperatures suitable for bacteria. A method for attaching Gram positive and negative bacteria on the surface of a normal Petri dish, showing a high reproducibly and a high cellular viability after 16 h. With these two key steps, an RT-CBA fit for microbiology is available.Next, to answer biological questions, intracellular interactions were explored by expression and validation of intracellular proteins with fluorescent tags suitable for RT-CBAs. First, we used the subunit B from the Shiga toxin (STxB) as a model to understand different aspects about the internalization processes. RT-CBAs allowed to discovery new features of STxB binding and mechanism to deliver small molecules or small proteins into cancer cells. Then, for exploring intracellular interactions, insect cells were bioengineered for evaluating the ability of small molecules to internalize and bind to its target. Using Carbonic anhydrase II – sulfonamides as a model system, the molecular interaction in the cytoplasm could be measured using a quencher label approach. The development of this kind of novel RT-CBA tools provide new information about drug candidates for targets that are not properly expressed in bacterial cells.The assays in this project can make drug design more efficient. Furthermore, the evaluation of binding activity of the new compounds developed by AEGIS, focusing on rare/neglected diseases, in a biological environment has the potential to accelerate drug discovery for the targeted emerging diseases.
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| 8. |
- Fabini, Edoardo, et al.
(författare)
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Monitoring drug-serum protein interactions for early ADME prediction through Surface Plasmon Resonance technology
- 2017
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Ingår i: Journal of Pharmaceutical and Biomedical Analysis. - : Elsevier BV. - 0731-7085 .- 1873-264X. ; 144, s. 188-194
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Tidskriftsartikel (refereegranskat)abstract
- Many molecules fail to reach the market due to poor pharmacokinetic (PK) properties, rendering the potential drug virtually unavailable for the primary target despite efficient administration to the body. PK properties of endogenous and exogenous compounds in mammals are dependent, among other factors, on their ability to interact with serum proteins. The extent of binding can greatly influence their ADME (adsorption, distribution, metabolism and execration) profile. Reliable and cost-effective bioavailability studies, early in the drug discovery process, can lead to an improvement of the success rate for compounds entering clinical trials. Optical biosensors based on surface plasmon resonance (SPR) detection emerged as an efficient approach to obtain large amounts of information about the binding of small molecules to serum proteins. Simple, automated and fast assays provide a good throughput, versatility and highly informative data output, rendering the methodology particularly suited for early screening. The ability to provide basic information on PK can be easily coupled to structure-activity relationship analysis. In this review, features of the technology and its employment for the study of serum protein-small molecule interactions are presented and discussed.
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| 9. |
- Fabini, Edoardo, et al.
(författare)
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Unveiling the Biochemistry of the Epigenetic Regulator SMYD3
- 2019
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Ingår i: Biochemistry. - : AMER CHEMICAL SOC. - 0006-2960 .- 1520-4995. ; 58:35, s. 3634-3645
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Tidskriftsartikel (refereegranskat)abstract
- SET and MYND domain-containing protein 3 (SMYD3) is a lysine methyltransferase that plays a central role in a variety of cancer diseases, exerting its pro-oncogenic activity by methylation of key proteins, of both nuclear and cytoplasmic nature. However, the role of SMYD3 in the initiation and progression of cancer is not yet fully understood and further biochemical characterization is required to support the discovery of therapeutics targeting this enzyme. We have therefore developed robust protocols for production, handling, and crystallization of SMYD3 and biophysical and biochemical assays for clarification of SMYD3 biochemistry and identification of useful lead compounds. Specifically, a time-resolved biosensor assay was developed for kinetic characterization of SMYD3 interactions. Functional differences in SMYD3 interactions with its natural small molecule ligands SAM and SAH were revealed, with SAM forming a very stable complex. A variety of peptides mimicking putative substrates of SMYD3 were explored in order to expose structural features important for recognition. The interaction between SMYD3 and some peptides was influenced by SAM. A nonradioactive SMYD3 activity assay using liquid chromatography-mass spectrometry (LC-MS) analysis explored substrate features of importance also for methylation. Methylation was notable only toward MAP kinase kinase kinase 2 (MAP3K2_K-260)-mimicking peptides, although binary and tertiary complexes were detected also with other peptides. The analysis supported a random bi-bi mechanistic model for SMYD3 methyltransferase catalysis. Our work unveiled complexities in SMYD3 biochemistry and resulted in procedures suitable for further studies and identification of novel starting points for design of effective and specific leads for this potential oncology target.
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| 10. |
- Huang, Hsin-Ho, et al.
(författare)
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Analysis of the leakage of gene repression by an artificial TetR-regulated promoter in cyanobacteria
- 2015
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Ingår i: BMC Research Notes. - : Springer Science and Business Media LLC. - 1756-0500. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: There is a need for strong and tightly regulated promoters to construct more reliable and predictable genetic modules for synthetic biology and metabolic engineering. For this reason we have previously constructed a TetR regulated L promoter library for the cyanobacterium Synechocystis PCC 6803. In addition to the L03 promoter showing wide dynamic range of transcriptional regulation, we observed the L09 promoter as unique in high leaky gene expression under repressed conditions. In the present study, we attempted to identify the cause of L09 promoter leakage. TetR binding to the promoter was studied by theoretical simulations of DNA breathing dynamics and by surface plasmon resonance (SPR) biosensor technology to analyze the kinetics of the DNA-protein interactions.RESULTS: DNA breathing dynamics of a promoter was computed with the extended nonlinear Peyrard-Bishop-Dauxois mesoscopic model to yield a DNA opening probability profile at a single nucleotide resolution. The L09 promoter was compared to the L10, L11, and L12 promoters that were point-mutated and different in repressed promoter strength. The difference between DNA opening probability profiles is trivial on the TetR binding site. Furthermore, the kinetic rate constants of TetR binding, as measured by SPR biosensor technology, to the respective promoters are practically identical. This suggests that a trivial difference in probability as low as 1 × 10(-4) cannot lead to detectable variations in the DNA-protein interactions. Higher probability at the downstream region of transcription start site of the L09 promoter compared to the L10, L11, and L12 promoters was observed. Having practically the same kinetics of binding to TetR, the leakage problem of the L09 promoter might be due to enhanced RNA Polymerase (RNAP)-promoter interactions in the downstream region.CONCLUSIONS: Both theoretical and experimental analyses of the L09 promoter's leakage problem exclude a mechanism of reduced TetR binding but instead suggest enhanced RNAP binding. These results assist in creating more tightly regulated promoters for realizing synthetic biology and metabolic engineering in biotechnological applications.
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