| 1. |
- Andersson, H.O., et al.
(author)
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Optimization of P1-P3 groups in symmetric and asymmetric HIV-1 protease inhibitors
- 2003
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In: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 270:8, s. 1746-1758
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Journal article (peer-reviewed)abstract
- HIV-1 protease is an important target for treatment of AIDS, and efficient drugs have been developed. However, the resistance and negative side effects of the current drugs has necessitated the development of new compounds with different binding patterns. In this study, nine C-terminally duplicated HIV-1 protease inhibitors were cocrystallised with the enzyme, the crystal structures analysed at 1.8-2.3 Å resolution, and the inhibitory activity of the compounds characterized in order to evaluate the effects of the individual modifications. These compounds comprise two central hydroxy groups that mimic the geminal hydroxy groups of a cleavage-reaction intermediate. One of the hydroxy groups is located between the d-oxygen atoms of the two catalytic aspartic acid residues, and the other in the gauche position relative to the first. The asymmetric binding of the two central inhibitory hydroxyls induced a small deviation from exact C2 symmetry in the whole enzyme-inhibitor complex. The study shows that the protease molecule could accommodate its structure to different sizes of the P2/P2' groups. The structural alterations were, however, relatively conservative and limited. The binding capacity of the S3/S3' sites was exploited by elongation of the compounds with groups in the P3/P3' positions or by extension of the P1/P1' groups. Furthermore, water molecules were shown to be important binding links between the protease and the inhibitors. This study produced a number of inhibitors with Ki values in the 100 picomolar range.
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| 2. |
- Elinder, Malin, et al.
(author)
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Experimental Validation of a Fragment Library for Lead Discovery Using SPR Biosensor Technology
- 2011
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In: Journal of Biomolecular Screening. - : Elsevier BV. - 1087-0571 .- 1552-454X. ; 16:1, s. 15-25
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Journal article (peer-reviewed)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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| 3. |
- Elinder, Malin, 1977-, et al.
(author)
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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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In: Journal of Biomolecular Screening. - : SAGE. - 1087-0571 .- 1552-454X. ; 14:4, s. 395-403
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Journal article (peer-reviewed)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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| 4. |
- Lundquist, Anna, et al.
(author)
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Biotinylated lipid bilayer disks as model membranes for biosensor analyses
- 2010
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In: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 405:2, s. 153-159
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Journal article (peer-reviewed)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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| 5. |
- Nordström, Helena, et al.
(author)
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Identification of MMP-12 Inhibitors by Using Biosensor-Based Screening of a Fragment Library
- 2008
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In: Journal of Medicinal Chemistry. - : American Chemical Society (ACS). - 0022-2623 .- 1520-4804. ; 51:12, s. 3449-3459
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Journal article (peer-reviewed)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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| 6. |
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| 7. |
- Abdurakhmanov, Eldar, 1977-, et al.
(author)
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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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In: Viruses. - : MDPI AG. - 1999-4915. ; 9:6
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Journal article (peer-reviewed)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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| 8. |
- Ahlsén, Göran, et al.
(author)
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Resistance profiles of cyclic and linear inhibitors of HIV-1 protease
- 2002
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In: Antiviral Chemistry & Chemotherapy. - 0956-3202 .- 2040-2066. ; 13:1, s. 27-37
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Journal article (peer-reviewed)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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| 9. |
- Al-Amin, Rasel A., Researcher, 1983-, et al.
(author)
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Monitoring drug–target interactions through target engagement-mediated amplification on arrays and in situ
- 2022
-
In: Nucleic Acids Research. - : Oxford University Press. - 0305-1048 .- 1362-4962. ; 50:22, s. e129-e129
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Journal article (peer-reviewed)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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| 10. |
- Al-Amin, Rasel Abdullah, Researcher, 1983-, et al.
(author)
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Sensitive protein detection using site-specifically oligonucleotide-conjugated nanobody reagents
- 2022
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In: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 98:28, s. 10054-10061
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Journal article (peer-reviewed)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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