| 1. |
- Elinder, Malin, et al.
(författare)
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Experimental Validation of a Fragment Library for Lead Discovery Using SPR Biosensor Technology
- 2011
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Ingår i: Journal of Biomolecular Screening. - : Elsevier BV. - 1087-0571 .- 1552-454X. ; 16:1, s. 15-25
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Tidskriftsartikel (refereegranskat)abstract
- A new fragment library for lead discovery has been designed and experimentally validated for use in surface plasmon resonance (SPR) biosensor-based screening. The 930 compounds in the library were selected from 4.6 million commercially available compounds using a series of physicochemical and medicinal chemistry filters. They were screened against 3 prototypical drug targets: HIV-1 protease, thrombin and carbonic anhydrase, and a nontarget: human serum albumin. compound solubility was not a problem under the conditions used for screening. The high sensitivity of the sensor surfaces allowed the detection of interactions for 35% to 97% of the fragments, depending on the target protein. None of the fragments was promiscuous (i.e., interacted with a stoichiometry ≥5:1 with all 4 proteins), and only 2 compounds dissociated slowly from all 4 proteins. The use of several targets proved valuable since several compounds would have been disqualified from the library on the grounds of promiscuity if fewer target proteins had been used. The experimental procedure allowed an efficient evaluation and exploration of the new fragment library and confirmed that the new library is suitable for SPR biosensor-based screening.
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| 2. |
- Elinder, Malin, 1977-, et al.
(författare)
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Screening for NNRTIs with Slow Dissociation and High Affinity for a Panel of HIV-1 RT Variants
- 2009
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Ingår i: Journal of Biomolecular Screening. - : SAGE. - 1087-0571 .- 1552-454X. ; 14:4, s. 395-403
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Tidskriftsartikel (refereegranskat)abstract
- A lead optimization library consisting of 800 HIV-1 nonnucleoside reverse transcriptase inhibitors (NNRTIs) was screened in parallel against 4 clinically relevant variants of HIV-1 RT (Wt, L100I, Y181C, and K103N) using a surface plasmon resonance-based biosensor. the aim was to identify inhibitors suitable in specific topical microbicides efficient for preventing the transmission of a range of clinically significant strains of HIV-1. the authors hypothesized that such compounds should have high affinity and slow dissociation rates for multiple variants of the target. to efficiently analyze the large amount of real-time data (sensorgrams) that were generated in the screening, they initially used signals from 3 selected time points to identify compounds with high affinity and slow dissociation for the complete panel of enzyme variants. hits were confirmed by visually inspecting the complete sensorgrams. two structurally unrelated compounds fulfilled the hit criteria, but only 1 compound was found to (a) compete with a known NNRTI for binding to the NNRTI site, (b) inhibit HIV-1 RT activity, and (c) inhibit HIV-1 replication in cell culture, for all 4 enzyme variants. this novel screening methodology offers high-resolution real-time kinetic data for multiple targets in parallel. it is expected to have broad applicability for the discovery of compounds with defined kinetic profiles, crucial for optimal therapeutic effects.
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| 3. |
- Gossas, Thomas, et al.
(författare)
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The advantage of biosensor analysis over enzyme inhibition studies for slow dissociating inhibitors : characterization of hydroxamate-based matrix metalloproteinase-12 inhibitors
- 2013
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Ingår i: MedChemComm. - : Royal Society of Chemistry (RSC). - 2040-2503 .- 2040-2511. ; 4:2, s. 432-442
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Tidskriftsartikel (refereegranskat)abstract
- The kinetic characteristics of hydroxamate-based inhibitors of matrix metalloproteinase (MMP)-12 were explored using an SPR biosensor-based assay and enzyme inhibition analysis. These high-affinity inhibitors were shown to dissociate very slowly from the enzyme-inhibitor complex while a carboxylate analogue had a much faster dissociation rate, verifying the importance of the hydroxamate group for the slow dissociation. Progress curve enzyme inhibition analysis confirmed that the hydroxamate compounds but not the carboxylate compound acted as time-dependent inhibitors. The slow dissociation excluded steady-state estimation of IC50-values and K-i values but also made K-i values from progress curve analysis unreliable. Although a full characterization of the inhibitors using biosensor analysis was limited by slow dissociation, it provided kinetic and mechanistic information of relevance for MMP drug discovery and avoided some pitfalls of conventional enzyme inhibition assays.
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| 4. |
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| 5. |
- Lundquist, Anna, et al.
(författare)
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Biotinylated lipid bilayer disks as model membranes for biosensor analyses
- 2010
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Ingår i: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 405:2, s. 153-159
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Tidskriftsartikel (refereegranskat)abstract
- The aim of this study was to investigate the potential of polyethylene glycol (PEG)-stabilized lipid bilayer disks as model membranes for surface plasmon resonance (SPR)-based biosensor analyses. Nanosized bilayer disks that included 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)(2000)] (DSPE-PEG(2000)-biotin) were prepared and structurally characterized by cryo-transmission electron microscopy (cryo-TEM) imaging. The biotinylated disks were immobilized via streptavidin to three different types of sensor chips (CM3, CM4, and CM5) varying in their degree of carboxymethylation and thickness of the dextran matrix. The bilayer disks were found to interact with and bind stably to the streptavidin-coated sensor surfaces. As a first step toward the use of these bilayer disks as model membranes in SPR-based studies of membrane proteins, initial investigations were carried out with cyclooxygenases 1 and 2 (COX 1 and COX 2). Bilayer disks were preincubated with the respective protein and thereafter allowed to interact with the sensor surface. The signal resulting from the interaction was, in both cases, significantly enhanced as compared with the signal obtained when disks alone were injected over the surface. The results of the study suggest that bilayer disks constitute a new and promising type of model membranes for SPR-based biosensor studies.
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| 6. |
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| 7. |
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| 8. |
- Nordström, Helena, 1974-
(författare)
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Fragment Based Drug Discovery with Surface Plasmon Resonance Technology
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Fragment based drug discovery (FBDD) has been applied to two protease drug targets, MMP-12 and HIV-1 protease. The primary screening and characterization of hit fragments were performed with surface plasmon resonance -technology. Further evaluation of the interaction was done by inhibition studies and in one case with X-ray crystallography. The focus of the two projects was different.Many MMP inhibitors contain a strong zinc chelating group, hydroxamate, interacting with the catalytic zinc atom. This strategy may be the cause for the low specificity of MMP inhibitors. Using FBDD we found a fragment with an unusual strong affinity for MMP-12. An inhibition assay confirmed that it was an inhibitor but indicated a stoichiometry of 2:1. Crystallography data revealed that an adduct of the fragment was bound in the active site, with interactions both with the catalytic zinc and the S1’ pocket. This may present a new scaffold for MMP-12 inhibitors.For HIV-1 protease the focus was on identifying inhibitors not sensitive to current resistance mutations. A fragment library for screening with SPR-technology was designed and used for screening against wild type enzyme and three variants with resistance mutations. Many of the hits were promiscuous but a number of fragments with possible allosteric inhibition mechanism were identified.The temperature dependency of the dissociation rate and reported resistance mutations was studied with thermodynamics. A good, but not perfect correlation was found between resistance and both the dissociation data and the free energy for dissociation compared to data from wild type enzyme. However, the type of mutation also influenced the results. The flap mutation G48V displayed thermodynamic profiles not completely correlating with resistance. It was found that dissociation rate and thermodynamics may complement each other when studying resistance, but only one of them may not be enough.
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| 9. |
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| 10. |
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| 11. |
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| 12. |
- Nordström, Helena, et al.
(författare)
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Identification of MMP-12 Inhibitors by Using Biosensor-Based Screening of a Fragment Library
- 2008
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Ingår i: Journal of Medicinal Chemistry. - : American Chemical Society (ACS). - 0022-2623 .- 1520-4804. ; 51:12, s. 3449-3459
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Tidskriftsartikel (refereegranskat)abstract
- Small inhibitors of matrix metalloproteinase 12 (MMP-12) have been identified with a biosensor-based screening strategy and a specifically designed fragment library. The interaction between fragments and three variants of the target and a reference protein with an active-site zinc ion was measured continuously by surface plasmon resonance. The developed experimental design overcame the inherent instability of MMP-12 and allowed the identification of fragments that interacted specifically with the active-site of MMP-12 but not with the reference protein. The interaction with MMP-12 for selected compounds were analyzed for concentration dependence and saturability. Compounds interacting distinctly with the target were further evaluated by an activity-based assay, verifying MMP-12 inhibition. Two effective inhibitors were identified, and the compound with highest affinity was confirmed to be a competitive inhibitor with an IC50 of 290 nM and a ligand efficiency of 0.7 kcal/mol heavy atom. This procedure integrates selectivity and binding site identification into the screening procedure and does not require structure determination.
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| 13. |
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| 14. |
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| 15. |
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| 16. |
- 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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| 17. |
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| 18. |
- Abdurakhmanov, Eldar, 1977-
(författare)
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Discovery and evaluation of direct acting antivirals against hepatitis C virus
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Until recently, the standard therapy for hepatitis C treatment has been interferon and ribavirin. Such treatment has only 50% efficacy and is not well tolerated. The emergence of new drugs has increased the treatment efficacy to 90%. Despite such an achievement, the success is limited since the virus mutates rapidly, causing the emergence of drug resistant forms. In addition, most new drugs were developed to treat genotype 1 infections. Thus, development of new potent antivirals is needed and drug discovery against hepatitis C is continued.In this thesis, a FRET-based protease assay was used to evaluate new pyrazinone based NS3 protease inhibitors that are structurally different to the newly approved and currently developing drugs. Several compounds in this series showed good potencies in the nanomolar range against NS3 proteases from genotype 1, 3, and the drug resistance variant R155K. We assume that these compounds can be further developed into drug candidates that possess activity against above mentioned enzyme variants.By using SPR technology, we analyzed interaction mechanisms and characteristics of allosteric inhibitors targeting NS5B polymerases from genotypes 1 and 3. The compounds exhibited different binding mechanisms and displayed a low affinity against NS5B from genotype 3.In order to evaluate the activity and inhibitors of the NS5B polymerase, we established an SPR based assay, which enables the monitoring of polymerization and its inhibition in real time. This assay can readily be implemented for the discovery of inhibitors targeting HCV.An SPR based fragment screening approach has also been established. A screen of a fragment library has been performed in order to identify novel scaffolds that can be used as a starting point for development of new allosteric inhibitors against NS5B polymerase. Selected fragments will be further elaborated to generate a new potent allosteric drug candidate.Alternative approaches have successfully been developed and implemented to the discovery of potential lead compounds targeting two important HCV drug targets.
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| 19. |
- Ahlsén, Göran, et al.
(författare)
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Resistance profiles of cyclic and linear inhibitors of HIV-1 protease
- 2002
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Ingår i: Antiviral Chemistry & Chemotherapy. - 0956-3202 .- 2040-2066. ; 13:1, s. 27-37
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Tidskriftsartikel (refereegranskat)abstract
- Resistance to anti-HIV protease drugs is a major problem in the design of AIDS drugs with long-term efficacy. To identify structural features associated with a certain resistance profile, the inhibitory properties of a series of symmetric and asymmetric cyclic sulfamide, cyclic urea and linear transition-state analogue inhibitors of HIV-1 protease were investigated using wild-type and mutant enzyme. To allow a detailed structure-inhibition analysis, enzyme with single, double, triple and quadruple combinations of G48V, V82A, 184V and L90M substitutions was used. Kinetic analysis of the mutants revealed that catalytic efficiency was 1-30% of that for the wild-type enzyme, a consequence of reduced kcat in all cases and an increased KM for all mutants except for the G48V enzyme. The overall structure-inhibitory profiles of the cyclic compounds were similar, and the inhibition of the V82A, 184V and G48V/L90M mutants were less efficient than of the wild-type enzyme. The greatest increase in Ki was generally observed for the 184V mutant and least for the G48V/L90M mutant, and additional combinations of mutations did not result in improved inhibition profiles for the cyclic compounds. An extended analysis of additional mutants, and including a set of linear compounds, showed that the profile was unique for each compound, and did not reveal any general structural features associated with a certain inhibition profile. The effects of structural modifications in the inhibitors, or of mutations, were not additive and they differed depending on their context. The results demonstrate the difficulties in predicting resistance, even for closely related compounds, and designing compounds with improved resistance profiles.
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| 20. |
- Al-Amin, Rasel A., Researcher, 1983-, et al.
(författare)
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Monitoring drug–target interactions through target engagement-mediated amplification on arrays and in situ
- 2022
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Ingår i: Nucleic Acids Research. - : Oxford University Press. - 0305-1048 .- 1362-4962. ; 50:22, s. e129-e129
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Tidskriftsartikel (refereegranskat)abstract
- Drugs are designed to bind their target proteins in physiologically relevant tissues and organs to modulate biological functions and elicit desirable clinical outcomes. Information about target engagement at cellular and subcellular resolution is therefore critical for guiding compound optimization in drug discovery, and for probing resistance mechanisms to targeted therapies in clinical samples. We describe a target engagement-mediated amplification (TEMA) technology, where oligonucleotide-conjugated drugs are used to visualize and measure target engagement in situ, amplified via rolling-circle replication of circularized oligonucleotide probes. We illustrate the TEMA technique using dasatinib and gefitinib, two kinase inhibitors with distinct selectivity profiles. In vitro binding by the dasatinib probe to arrays of displayed proteins accurately reproduced known selectivity profiles, while their differential binding to fixed adherent cells agreed with expectations from expression profiles of the cells. We also introduce a proximity ligation variant of TEMA to selectively investigate binding to specific target proteins of interest. This form of the assay serves to improve resolution of binding to on- and off-target proteins. In conclusion, TEMA has the potential to aid in drug development and clinical routine by conferring valuable insights in drug–target interactions at spatial resolution in protein arrays, cells and in tissues.
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| 21. |
- Al-Amin, Rasel Abdullah, Researcher, 1983-, et al.
(författare)
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Sensitive protein detection using site-specifically oligonucleotide-conjugated nanobody reagents
- 2022
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 98:28, s. 10054-10061
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Tidskriftsartikel (refereegranskat)abstract
- High-quality affinity probes are critical for sensitive and specific protein detection, in particular for detection of protein biomarkers in the early phases of disease development. Proximity extension assays (PEAs) have been used for high-throughput multiplexed protein detection of up to a few thousand different proteins in one or a few microliters of plasma. Clonal affinity reagents can offer advantages over the commonly used polyclonal antibodies (pAbs) in terms of reproducibility and standardization of such assays. Here, we explore nanobodies (Nbs) as an alternative to pAbs as affinity reagents for PEA. We describe an efficient site-specific approach for preparing high-quality oligo-conjugated Nb probes via enzyme coupling using Sortase A (SrtA). The procedure allows convenient removal of unconjugated affinity reagents after conjugation. The purified high-grade Nb probes were used in PEA, and the reactions provided an efficient means to select optimal pairs of binding reagents from a group of affinity reagents. We demonstrate that Nb-based PEA (nano-PEA) for interleukin-6 (IL6) detection can augment assay performance, compared to the use of pAb probes. We identify and validate Nb combinations capable of binding in pairs without competition for IL6 antigen detection by PEA.
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| 22. |
- Al-Amin, Rasel A., 1983-, et al.
(författare)
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Target Engagement-Mediated Amplification for Monitoring Drug-Target Interactions in Situ
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- It is important to determine the localization of drugs or drug candidates at cellular and subcellular resolution in relevant clinical specimens. This is necessary to evaluate drug candidates from early stages of drug development to clinical evaluation of mutations potentially causing resistance to targeted therapy. We describe a technology where oligonucleotide-conjugated drug molecules are used to visualize and measure target engagement in situ via rolling-circle amplification (RCA) of circularized oligonucleotide probes (padlock probes). We established this target engagement-mediated amplification (TEMA) technique using kinase inhibitor precursor compounds, and we applied the assay to investigate target interactions by microscopy in pathology tissue sections and using flow cytometry for blood samples from patients, as well as in commercial arrays including almost half of all human proteins. In the variant proxTEMAtechnique, in situ proximity ligation assays were performed by combining drug-DNA conjugates with antibody-DNA conjugates to specifically reveal drug binding to particular on- or off-targets in pathological tissues sections. In conclusion, the TEMA methods successfully visualize drug-target interaction by experimental and clinically approved kinase inhibitors in situ and with kinases among a large collection of arrayed proteins.
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| 23. |
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| 24. |
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| 25. |
- Alterman, Mathias, et al.
(författare)
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P1/P1' modified HIV protease inhibitors as tools in two new sensitive surface plasmon resonance biosensor screening assays
- 2001
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Ingår i: European Journal of Pharmaceutical Sciences. - : Elsevier. - 0928-0987 .- 1879-0720. ; 13:2, s. 203-212
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Tidskriftsartikel (refereegranskat)abstract
- The commonly used HIV-1 protease assays rely on measurements of the effect of inhibitions on the hydrolysis rate of synthetic peptides. Recently an assay based on surface plasmon resonance (SPR) was introduced. We have taken advantage of the fact that the SPR signal is proportional to the mass of the analyte interacting with the immobilised molecule and developed two new improved efficient competition assay methods. Thus, high molecular weight binders were used as amplifiers of the surface plasmon resonance signal. Linkers were attached by a Heck reaction to the para-positions of the P1/P1′ benzyloxy groups of a linear C2-symmetric C-terminal duplicated inhibitor to enable (a) biotin labelling or (b) direct immobilisation of the inhibitor to the biosensor surface matrix. The interaction properties of a series of 17 structurally diverse inhibitors was assessed and compared to previously reported data. The most sensitive assay was obtained by immobilising the enzyme and amplifying the signal with an antibody, giving a detection range between 0.1 nM and 10 μM. Immobilisation of the inhibitor resulted in a stable and durable surface but a narrower detection range (1–100 nM). The two competition assays are anticipated to be very suitable for fast screening of potential HIV inhibitors.
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