10 research outputs found
Complex formation between albumin and long-acting insulin analogues:A Small-Angle X-ray Scattering and Molecular Dynamics Study
No full textThe use of biopharmaceuticals in the treatment of diseases such as diabetes, cancer, and hemophilia has increased dramatically over the past decades. Despite their many advantages such as high potency, specificity, and low toxicity, many biopharmaceuticals suffer from inherent chemical and physical instabilities and short plasma half-lives, which make their formulation development and delivery challenging. Lipidation is a successful strategy for extending the half-lives of peptide drugs through lipidation-induced selfassociation and association to albumin. Though albumin association is exploited by several approved lipidated peptide drugs, structural knowledge about the albumin-peptide complexes formed and their interactions on the atomic level is limited. This thesis aims to shed light on self-association and albumin-association of two lipidated insulin analogues, insulin detemir and insulin degludec, through an interdisciplinary approach using smallangle X-ray scattering (SAXS) and molecular dynamics (MD) simulations.We succeeded in modelling the solution structures of a detemir trihexamer, and albumininsulin analogue complexes in 1:6, 1:12, and 2:12 stoichiometries based on SAXS data, and proposed equilibria for albumin-detemir and albumin-degludec mixtures. The structures are the first detemir trihexamer structure and the first structures of complexes between albumin and lipidated insulin analogues, and contribute to an understanding of detemir and degludec’s prolonged actions. The albumin-detemir hexamer solution structure is ambiguous and shows four possible detemir binding sites. In order to determine the most favorable binding site and obtain knowledge on the specific interactions in the complex, these binding sites were investigated by MD simulations and molecular mechanics Poisson-Boltzmann surface area freeenergy calculations. The overlapping FA3-FA4 binding site on albumin was found to be the most favorable detemir binding site, and two lipidated detemir residues were found to contribute to the binding with favorable electrostatic and van der Waals interactions. The atomic-level insights on the albumin-detemir binding could be utilized in a more rational design of future lipidated peptide drugs. The study, furthermore, highlights the strength of combining SAXS with MD simulations. The effect of albumin-detemir association on detemir’s stability was explored through different stress tests to investigate whether albumin-association could potentially be utilized in a formulation perspective. The presence of albumin was found to enhance detemir’s stability against freeze-thaw and agitation stresses almost independently on complex formation, suggesting that albumin-detemir complex formation does not lead to further stabilization
Investigations of albumin-insulin detemir complexes using molecular dynamics simulations and free energy calculations
No full textInsulin detemir is a lipidated insulin analogue that obtains a half-life extension by oligomerization and reversible binding to human serum albumin. In the present study, the complex between a detemir hexamer and albumin is investigated by an integrative approach combining molecular dynamics (MD) simulations, molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) free energy calculations and dynamic light scattering (DLS) experiments. Recent reported small angle X-ray scattering data could not unambiguously resolve the exact binding site of detemir on albumin. We therefore applied MD simulations to deduce the binding site and key protein-protein interactions. MD simulations were started from initial complex structures based on the SAXS models and free energies of binding were estimated from the simulations by using the MM-PBSA approach for the different binding positions. The results suggest that the overlapping FA3-FA4 binding site (named FA4) is the most favorable site with a calculated free energy of binding of -28±6 kcal/mol and a good fit to the reported SAXS data throughout the simulations. Multiple salt bridges, hydrogen bonds and favorable van der Waals interactions are observed in the binding interface that promote complexation. The binding to FA4 is further supported by DLS competition experiments with the prototypical FA4 ligand, ibuprofen, showing displacement of detemir by ibuprofen. This study provides information on albumin-detemir binding on a molecular level, which could be utilized in a rational design of future lipidated albumin-binding peptides
Solution structures of long-acting insulin analogues and their complexes with albumin
No full textThe lipidation of peptide drugs is one strategy to obtain extended half-lives, enabling once-daily or even less frequent injections for patients. The half-life extension results from a combination of self-association and association with human serum albumin (albumin). The self-association and association with albumin of two insulin analogues, insulin detemir and insulin degludec, were investigated by small-angle X-ray scattering (SAXS) and dynamic light scattering (DLS) in phenolic buffers. Detemir shows concentration-dependent self-association, with an equilibrium between hexamer, dihexamer, trihexamer and larger species, while degludec appears as a dihexamer independent of concentration. The solution structure of the detemir trihexamer has a bent shape. The stoichiometry of the association with albumin was studied using DLS. For albumin-detemir the molar stoichiometry was determined to be 1:6 (albumin:detemir ratio) and for albumin-degludec it was between 1:6 and 1:12 (albumin:degludec ratio). Batch SAXS measurements of a 1:6 albumin:detemir concentration series revealed a concentration dependence of complex formation. The data allowed the modelling of a complex between albumin and a detemir hexamer and a complex consisting of two albumins binding to opposite ends of a detemir dihexamer. Measurements of size-exclusion chromatography coupled to SAXS revealed a complex between a degludec dihexamer and albumin. Based on the results, equilibria for the albumin-detemir and albumin-degludec mixtures are proposed
Substitutions in SARS-CoV-2 Mpro Selected by Protease Inhibitor Boceprevir Confer Resistance to Nirmatrelvir
No full textNirmatrelvir, which targets the SARS-CoV-2 main protease (Mpro), is the first-in-line drug for prevention and treatment of severe COVID-19, and additional Mpro inhibitors are in development. However, the risk of resistance development threatens the future efficacy of such direct-acting antivirals. To gain knowledge on viral correlates of resistance to Mpro inhibitors, we selected resistant SARS-CoV-2 under treatment with the nirmatrelvir-related protease inhibitor boceprevir. SARS-CoV-2 selected during five escape experiments in VeroE6 cells showed cross-resistance to nirmatrelvir with up to 7.3-fold increased half-maximal effective concentration compared to original SARS-CoV-2, determined in concentration–response experiments. Sequence analysis revealed that escape viruses harbored Mpro substitutions L50F and A173V. For reverse genetic studies, these substitutions were introduced into a cell-culture-infectious SARS-CoV-2 clone. Infectivity titration and analysis of genetic stability of cell-culture-derived engineered SARS-CoV-2 mutants showed that L50F rescued the fitness cost conferred by A173V. In the concentration–response experiments, A173V was the main driver of resistance to boceprevir and nirmatrelvir. Structural analysis of Mpro suggested that A173V can cause resistance by making boceprevir and nirmatrelvir binding less favorable. This study contributes to a comprehensive overview of the resistance profile of the first-in-line COVID-19 treatment nirmatrelvir and can thus inform population monitoring and contribute to pandemic preparedness
Neutralisation resistance of SARS-CoV-2 spike-variants is primarily mediated by synergistic receptor binding domain substitutions
No full textThe evolution of SARS-CoV-2 has led to the emergence of numerous variants of concern (VOCs), marked by changes in the viral spike glycoprotein, the primary target for neutralising antibody (nAb) responses. Emerging VOCs, particularly omicron sub-lineages, show resistance to nAbs induced by prior infection or vaccination. The precise spike protein changes contributing to this resistance remain unclear in infectious cell culture systems. In the present study, a large panel of infectious SARS-CoV-2 mutant viruses, each with spike protein changes found in VOCs, including omicron JN.1 and its derivatives KP.2 and KP.3, was generated using a reverse genetic system. The susceptibility of these viruses to antibody neutralisation was measured using plasma from convalescent and vaccinated individuals. Synergistic roles of combined substitutions in the spike receptor binding domain (RBD) were observed in neutralisation resistance. However, recombinant viruses with the entire spike protein from a specific VOC showed enhanced resistance, indicating that changes outside the RBD are also significant. In silico analyses of spike antibody epitopes suggested that changes in neutralisation could be due to altered antibody binding affinities. Assessing ACE2 usage for entry through anti-ACE2 antibody blocking and ACE2 siRNA revealed that omicron BA.2.86 and JN.1 mutant viruses were less dependent on ACE2 for entry. However, surface plasmon resonance analysis showed increased affinity for ACE2 for both BA.2.86 and JN.1 compared to the ancestral spike. This detailed analysis of specific changes in the SARS-CoV-2 spike enhances understanding of coronavirus evolution, particularly regarding neutralising antibody evasion and ACE2 entry receptor dependence
A systematic literature review and network meta-analysis of treatments for patients with untreated multiple myeloma not eligible for stem cell transplantation
No full textIn newly diagnosed multiple myeloma (MM), patients ineligible for front-line autologous stem cell transplantation (ASCT), melphalan and prednisone (MP) with thalidomide (MPT) or bortezomib (VMP) are standard first-line therapeutic options. Despite new treatment regimens incorporating bortezomib or lenalidomide, MM remains incurable. The FIRST study demonstrated significant improvement in progression-free survival (PFS) and overall survival (OS) for the combination of lenalidomide and low-dose dexamethasone (Rd) until progression vs. MPT in transplant-ineligible ndMM patients. However, to date no head-to-head randomized controlled trials (RCTs) have compared Rd or MPT versus VMP. We conducted a network meta-analysis using RCTs identified through a systematic literature review to evaluate the relative efficacy of Rd versus other regimens on survival endpoints in previously untreated MM patients ineligible for ASCT. In this analysis, Rd was associated with a significant PFS and survival advantage versus other first-line treatments (VMP, MPT, MP), challenging the role of alkylators in this setting. © 2016 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group
SARS-CoV-2 Remdesivir Exposure Leads to Different Evolutionary Pathways That Converge in Moderate Levels of Drug Resistance
No full textVarious SARS-CoV-2 remdesivir resistance-associated substitutions (RAS) have been reported, but a comprehensive comparison of their resistance levels is lacking. We identified novel RAS and performed head-to-head comparisons with known RAS in Vero E6 cells. A remdesivir escape polyclonal virus exhibited a 3.6-fold increase in remdesivir EC50 and mutations throughout the genome, including substitutions in nsp12 (E796D) and nsp14 (A255S). However, in reverse-genetics infectious assays, viruses harboring both these substitutions exhibited only a slight decrease in remdesivir susceptibility (1.3-fold increase in EC50). The nsp12-E796D substitution did not impair viral fitness (Vero E6 cells or Syrian hamsters) and was reported in a remdesivir-treated COVID-19 patient. In replication assays, a subgenomic replicon containing nsp12-E796D+nsp14-A255S led to a 16.1-fold increase in replication under remdesivir treatment. A comparison with known RAS showed that S759A, located in the active site of nsp12, conferred the highest remdesivir resistance (106.1-fold increase in replication). Nsp12-RAS V166A/L, V792I, E796D or C799F, all adjacent to the active site, caused intermediate resistance (2.0- to 11.5-fold), whereas N198S, D484Y, or E802D, located farther from the active site, showed no resistance (≤2.0-fold). In conclusion, our classification system, correlating replication under remdesivir treatment with RAS location in nsp12, shows that most nsp12-RAS cause moderate resistance
A systematic literature review and network meta-analysis of treatments for patients with untreated multiple myeloma not eligible for stem cell transplantation.
No full textIn newly diagnosed multiple myeloma (MM), patients ineligible for front-line autologous stem cell transplantation (ASCT), melphalan and prednisone (MP) with thalidomide (MPT) or bortezomib (VMP) are standard first-line therapeutic options. Despite new treatment regimens incorporating bortezomib or lenalidomide, MM remains incurable. The FIRST study demonstrated significant improvement in progression-free survival (PFS) and overall survival (OS) for the combination of lenalidomide and low-dose dexamethasone (Rd) until progression vs. MPT in transplant-ineligible ndMM patients. However, to date no head-to-head randomized controlled trials (RCTs) have compared Rd or MPT versus VMP. We conducted a network meta-analysis using RCTs identified through a systematic literature review to evaluate the relative efficacy of Rd versus other regimens on survival endpoints in previously untreated MM patients ineligible for ASCT. In this analysis, Rd was associated with a significant PFS and survival advantage versus other first-line treatments (VMP, MPT, MP), challenging the role of alkylators in this setting
Multiple Myeloma Treatment in Real-world Clinical Practice: Results of a Prospective, Multinational, Noninterventional Study
No full textMultiple myeloma (MM) remains an incurable disease, with little information available on its management in real-world clinical practice. The results of the present prospective, noninterventional observational study revealed great diversity in the treatment regimens used to treat MM. Our results also provide data to inform health economic, pharmacoepidemiologic, and outcomes research, providing a framework for the design of protocols to improve the outcomes of patients with MM. Background: The present prospective, multinational, noninterventional study aimed to document and describe real-world treatment regimens and disease progression in multiple myeloma (MM) patients. Patients and Methods: Adult patients initiating any new MM therapy from October 2010 to October 2012 were eligible. A multistage patient/site recruitment model was applied to minimize the selection bias; enrollment was stratified by country, region, and practice type. The patient medical and disease features, treatment history, and remission status were recorded at baseline, and prospective data on treatment, efficacy, and safety were collected electronically every 3 months. Results: A total of 2358 patients were enrolled. Of these patients, 775 and 1583 did and did not undergo stem cell transplantation (SCT) at any time during treatment, respectively. Of the patients in the SCT and non-SCT groups, 49%, 21%, 14%, and 15% and 57%, 20%, 12% and 10% were enrolled at treatment line 1, 2, 3, and ≥ 4, respectively. In the SCT and non-SCT groups, 45% and 54% of the patients had received bortezomib-based therapy without thalidomide/lenalidomide, 12% and 18% had received thalidomide/lenalidomide-based therapy without bortezomib, and 30% and 4% had received bortezomib plus thalidomide/lenalidomide-based therapy as frontline treatment, respectively. The corresponding proportions of SCT and non-SCT patients in lines 2, 3, and ≥ 4 were 45% and 37%, 30% and 37%, and 12% and 3%, 33% and 27%, 35% and 32%, and 8% and 2%, and 27% and 27%, 27% and 23%, and 6% and 4%, respectively. In the SCT and non-SCT patients, the overall response rate was 86% to 97% and 64% to 85% in line 1, 74% to 78% and 59% to 68% in line 2, 55% to 83% and 48% to 60% in line 3, and 49% to 65% and 36% and 45% in line 4, respectively, for regimens that included bortezomib and/or thalidomide/lenalidomide. Conclusion: The results of our prospective study have revealed great diversity in the treatment regimens used to manage MM in real-life practice. This diversity was linked to factors such as novel agent accessibility and evolving treatment recommendations. Our results provide insight into associated clinical benefits. © 2018 The Author
Nirmatrelvir-resistant SARS-CoV-2 variants with high fitness in an infectious cell culture system
No full textThe oral protease inhibitor nirmatrelvir is of key importance for prevention of severe coronavirus disease 2019 (COVID-19). To facilitate resistance monitoring, we studied severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) escape from nirmatrelvir in cell culture. Resistant variants harbored combinations of substitutions in the SARS-CoV-2 main protease (Mpro). Reverse genetics revealed that E166V and L50F + E166V conferred high resistance in infectious culture, replicon, and Mpro systems. While L50F, E166V, and L50F + E166V decreased replication and Mpro activity, L50F and L50F + E166V variants had high fitness in the infectious system. Naturally occurring L50F compensated for fitness cost of E166V and promoted viral escape. Molecular dynamics simulations revealed that E166V and L50F + E166V weakened nirmatrelvir-Mpro binding. Polymerase inhibitor remdesivir and monoclonal antibody bebtelovimab retained activity against nirmatrelvir-resistant variants, and combination with nirmatrelvir enhanced treatment efficacy compared to individual compounds. These findings have implications for monitoring and ensuring treatments with efficacy against SARS-CoV-2 and emerging sarbecoviruses
