Comparison of the pH- and thermally-induced fluctuations of a therapeutic antibody Fab fragment by molecular dynamics simulation

Successful development of protein therapeutics depends critically on achieving stability under a range of conditions. A deeper understanding of the drivers of instability across different stress conditions, will enable the engineering of more robust protein scaffolds. We compared the impacts of low pH and high temperature stresses on the structure of a humanized antibody fragment (Fab) A33, using atomistic molecular dynamics simulations, using a recent 2.5 Å crystal structure. This revealed that low-pH induced the loss of native contacts in the domain CL. By contrast, thermal stress led to 5–7% loss of native contacts in all four domains, and simultaneous loss of >30% of native contacts in the VL-VH and CL-CH interfaces. This revealed divergent destabilising pathways under the two different stresses. The underlying cause of instability was probed using FoldX and Rosetta mutation analysis, and packing density calculations. These agreed that mutations in the CL domain, and CL-CH1 interface have the greatest potential for stabilisation of Fab A33. Several key salt bridge losses underpinned the conformational change in CL at low pH, whereas at high temperature, salt bridges became more dynamic, thus contributing to an overall destabilization. Lastly, the unfolding events at the two stress conditions exposed different predicted aggregation-prone regions (APR) to solvent, which would potentially lead to different aggregation mechanisms. Overall, our results identified the early stages of unfolding and stability-limiting regions of Fab A33, and the VH and CL domains as interesting future targets for engineering stability to both pH- and thermal-stresses simultaneously

Diverse cytomotive actins and tubulins share a polymerization switch mechanism conferring robust dynamics

Protein filaments are used in myriads of ways to organize other molecules within cells. Some filament-forming proteins couple the hydrolysis of nucleotides to their polymerization cycle, thus powering the movement of other molecules. These filaments are termed cytomotive. Only members of the actin and tubulin protein superfamilies are known to form cytomotive filaments. We examined the basis of cytomotivity via structural studies of the polymerization cycles of actin and tubulin homologs from across the tree of life. We analyzed published data and performed structural experiments designed to disentangle functional components of these complex filament systems. Our analysis demonstrates the existence of shared subunit polymerization switches among both cytomotive actins and tubulins, i.e., the conformation of subunits switches upon assembly into filaments. These cytomotive switches can explain filament robustness, by enabling the coupling of kinetic and structural polarities required for cytomotive behaviors and by ensuring that single cytomotive filaments do not fall apart

Crystal structures and molecular dynamics simulations of a humanised antibody fragment at acidic to basic pH

Antibody-fragment (Fab) therapy development has the potential to be accelerated by computational modelling and simulations that predict their target binding, stability, formulation, manufacturability, and the impact of further protein engineering. Such approaches are currently predicated on starting with good crystal structures that closely represent those found under the solution conditions to be simulated. A33 Fab, is an undeveloped immunotherapeutic antibody candidate that was targeted to the human A33 antigen homogeneously expressed in 95% cases of primary and metastatic colorectal cancers. It is now used as a very well characterised testing ground for developing analytics, formulation and protein engineering strategies, and to gain a deeper understanding of mechanisms of destabilisation, representative of the wider therapeutic Fab platform. In this article, we report the structure of A33 Fab in two different crystal forms obtained at acidic and basic pH. The structures overlapped with RMSD of 1.33 Å overall, yet only 0.5 Å and 0.76 Å for the variable- and constant regions alone. While most of the differences were within experimental error, the switch linker between the variable and the constant regions showed some small differences between the two pHs. The two structures then enabled a direct evaluation of the impact of initial crystal structure selection on the outcomes of molecular dynamics simulations under different conditions, and their subsequent use for determining best fit solution structures using previously obtained small-angle x-ray scattering (SAXS) data. The differences in the two structures did not have a major impact on MD simulations regardless of the pH, other than a slight persistence of structure affecting the solvent accessibility of one of the predicted APR regions of A33 Fab. Interestingly, despite being obtained at pH 4 and pH 9, the two crystal structures were more similar to the SAXS solution structures obtained at pH 7, than to those at pH 4 or pH 9. Furthermore, the P65 crystal structure from pH 4 was also a better representation of the solution structures at any other pH, than was the P1 structure obtained at pH 9. Thus, while obtained at different pH, the two crystal structures may represent highly (P65) and lesser (P1) populated species that both exist at pH 7 in solution. These results now lay the foundation for confident MD simulations of A33 Fab that rationalise or predict behaviours in a range of conditions

Emerging variants of SARS-CoV-2 NSP10 highlight strong functional conservation of its binding to two non-structural proteins, NSP14 and NSP16

The coronavirus SARS-CoV-2 protects its RNA from being recognized by host immune responses by methylation of its 5’ end, also known as capping. This process is carried out by two enzymes, non-structural protein 16 (NSP16) containing 2’-O-methyltransferase and NSP14 through its N7 methyltransferase activity, which are essential for the replication of the viral genome as well as evading the host’s innate immunity. NSP10 acts as a crucial cofactor and stimulator of NSP14 and NSP16. To further understand the role of NSP10, we carried out a comprehensive analysis of >13 million globally collected whole-genome sequences (WGS) of SARS-CoV-2 obtained from the Global Initiative Sharing All Influenza Data (GISAID) and compared it with the reference genome Wuhan/WIV04/2019 to identify all currently known variants in NSP10. T12I, T102I, and A104V in NSP10 have been identified as the three most frequent variants and characterized using X-ray crystallography, biophysical assays and enhanced sampling simulations. In contrast to other proteins such as spike and NSP6, NSP10 is significantly less prone to mutation due to its crucial role in replication. The functional effects of the variants were examined for their impact on the binding affinity and stability of both NSP14-NSP10 and NSP16-NSP10 complexes. These results highlight the limited changes induced by variant evolution in NSP10 and reflect on the critical roles NSP10 plays during the SARS-CoV-2 life cycle. These results also indicate that there is limited capacity for the virus to overcome inhibitors targeting NSP10 via the generation of variants in inhibitor binding pockets. eLife assessment This study presents a valuable finding on variations within the RNA synthesis protein of SARS-CoV-2, the virus that is responsible for COVID 19. The evidence supporting the claims of the authors is solid, although a more in-depth analysis of the structures and simulations would have strengthened the conclusions of the work. This work has implications for drug design and will be of broad interest to the general biophysics and structural biology community

Identification of fragments binding to SARS-CoV-2 nsp10 reveals ligand-binding sites in conserved interfaces between nsp10 and nsp14/nsp16

Since the emergence of SARS-CoV-2 in 2019, Covid-19 has developed into a serious threat to our health, social and economic systems. Although vaccines have been developed in a tour-de-force and are now increasingly available, repurposing of existing drugs has been less successful. There is a clear need to develop new drugs against SARS-CoV-2 that can also be used against future coronavirus infections. Non-structural protein 10 (nsp10) is a conserved stimulator of two enzymes crucial for viral replication, nsp14 and nsp16, exhibiting exoribonuclease and methyltransferase activities. Interfering with RNA proofreading or RNA cap formation represents intervention strategies to inhibit replication. We applied fragment-based screening using nano differential scanning fluorometry and X-ray crystallography to identify ligands targeting SARS-CoV-2 nsp10. We identified four fragments located in two distinct sites: one can be modelled to where it would be located in the nsp14–nsp10 complex interface and the other in the nsp16–nsp10 complex interface. Microscale thermophoresis (MST) experiments were used to quantify fragment affinities for nsp10. Additionally, we showed by MST that the interaction by nsp14 and 10 is weak and thereby that complex formation could be disrupted by small molecules. The fragments will serve as starting points for the development of more potent analogues using fragment growing techniques and structure-based drug design

Photocrosslinked Bioreducible Polymeric Nanoparticles for Enhanced Systemic siRNA Delivery as Cancer Therapy

Clinical translation of polymer‐based nanocarriers for systemic delivery of RNA has been limited due to poor colloidal stability in the blood stream and intracellular delivery of the RNA to the cytosol. To address these limitations, this study reports a new strategy incorporating photocrosslinking of bioreducible nanoparticles for improved stability extracellularly and rapid release of RNA intracellularly. In this design, the polymeric nanocarriers contain ester bonds for hydrolytic degradation and disulfide bonds for environmentally triggered small interfering RNA (siRNA) release in the cytosol. These photocrosslinked bioreducible nanoparticles (XbNPs) have a shielded surface charge, reduced adsorption of serum proteins, and enable superior siRNA‐mediated knockdown in both glioma and melanoma cells in high‐serum conditions compared to non‐crosslinked formulations. Mechanistically, XbNPs promote cellular uptake and the presence of secondary and tertiary amines enables efficient endosomal escape. Following systemic administration, XbNPs facilitate targeting of cancer cells and tissue‐mediated siRNA delivery beyond the liver, unlike conventional nanoparticle‐based delivery. These attributes of XbNPs facilitate robust siRNA‐mediated knockdown in vivo in melanoma tumors colonized in the lungs following systemic administration. Thus, biodegradable polymeric nanoparticles, via photocrosslinking, demonstrate extended colloidal stability and efficient delivery of RNA therapeutics under physiological conditions, and thereby potentially advance systemic delivery technologies for nucleic acid‐based therapeutics

Integrated care for patients with advanced chronic obstructive pulmonary disease

Przewlekła obturacyjna choroba płuc (POChP) jest trzecią przyczyną zgonów i trzecią przyczyną niesprawności (mierzonej za pomocą wskaźnika DALY) u osób powyżej 60. roku życia. Ciężką lub bardzo ciężką postać POChP (wskaźnik FEV1, odpowiednio, poniżej 50% i 30% należnej wartości) rozpoznaje się u mniej więcej jednej piątej ogółu chorych. Chorzy na zaawansowaną POChP z reguły cierpią z powodu fizycznej i umysłowej niepełnosprawności, w wielu przypadkach źle radzą sobie z problemami wynikającymi z choroby i współistniejących schorzeń. To z kolei stwarza duże ryzyko zaostrzeń POChP, które prowadzą do pogorszenia przebiegu choroby, istotnego zwiększenia kosztów medycznej opieki oraz zwiększenia ryzyka zgonu. Obecna organizacja medycznej opieki dla tej grupy chorych nie zapewnia odpowiednich warunków zdrowotnych i socjalnych. Wydaje się jednak, że zastosowanie modelu zintegrowanej opieki, zalecanego przez Światową Organizację Zdrowia stworzyłoby możliwość poprawy sytuacji chorych na zaawansowane POChP. W Polsce ta forma opieki jest realizowana od kilkunastu lat w zakładach medycyny paliatywnej i hospicjach w odniesieniu do chorych na zaawansowane nowotwory. Wykorzystanie tych doświadczeń powinno pomóc w integracji działań wokół lekarzy rodzinnych i wyspecjalizowanej pielęgniarki, z jednoczesnym zapewnieniem dostępu do specjalistów w zależności od indywidualnych potrzeb chorego. Powinno także umożliwić szersze wykorzystanie pracowników pomocniczych, jak: pracownicy socjalni, pomocnicy medyczni oraz wolontariusze, a także psycholodzy i duchowni (zwłaszcza w końcowej fazie choroby). Pneumonol. Alergol. Pol. 2010; 78, 2: 126-132Chronic obstructive pulmonary disease (COPD) is the third cause of mortality and disability (assed by DALY) among patients above 60 year old. Severe and very severe COPD (FEV1 = equal or less than 50% and 30% of expected value, respectively) is estimated at 20% of all COPD patients. Advanced COPD usually leads to physical and mental deterioration, the patients often manage with the problems caused by the disease and other comorbidities poorly. This leads to increased risk of COPD exacerbations and further deterioration of the patient’s status, increased costs of medical care and eventually increased risk of death. Current organization of medical care for those patients does not provide adequate health and social support for them. However, it seems that introducing an integrated approach proposed by World Health Organization, could improve the situation of advanced COPD patients. In Poland, this kind of care has been provided in advanced cancer patients throughout stationary palliative care units and hospices during the last several years. This experience should be helpful in integrating actions of general practitioners and specialized nurses, as well as providing access for the specialists’ consultations according to the individual needs of the patients. It should also allow for broad cooperation with auxiliary staff, such as social workers, medical assistants and volunteers, as well as psychologists and clergymen (especially in the terminal phase of the disease). Pneumonol. Alergol. Pol. 2010; 78, 2: 126-13

Activating Transcription Factor 4 Modulates TGFβ-Induced Aggressiveness in Triple-Negative Breast Cancer via SMAD2/3/4 and mTORC2 Signaling

Purpose: On the basis of the identified stress-independent cellular functions of activating transcription factor 4 (ATF4), we reported enhanced ATF4 levels in MCF10A cells treated with TGFβ1. ATF4 is overexpressed in patients with triple-negative breast cancer (TNBC), but its impact on patient survival and the underlying mechanisms remain unknown. We aimed to determine ATF4 effects on patients with breast cancer survival and TNBC aggressiveness, and the relationships between TGFβ and ATF4. Defining the signaling pathways may help us identify a cell signaling-tailored gene signature.Experimental Design: Patient survival data were determined by Kaplan-Meier analysis. Relationship between TGFβ and ATF4, their effects on aggressiveness (tumor proliferation, metastasis, and stemness), and the underlying pathways were analyzed in three TNBC cell lines and in vivo using patient-derived xenografts (PDX).Results: ATF4 overexpression correlated with TNBC patient survival decrease and a SMAD-dependent crosstalk between ATF4 and TGFβ was identified. ATF4 expression inhibition reduced migration, invasiveness, mammosphere-forming efficiency, proliferation, epithelial-mesenchymal transition, and antiapoptotic and stemness marker levels. In PDX models, ATF4 silencing decreased metastases, tumor growth, and relapse after chemotherapy. ATF4 was shown to be active downstream of SMAD2/3/4 and mTORC2, regulating TGFβ/SMAD and mTOR/RAC1-RHOA pathways independently of stress. We defined an eight-gene signature with prognostic potential, altered in 45% of 2,509 patients with breast cancer.Conclusions: ATF4 may represent a valuable prognostic biomarker and therapeutic target in patients with TNBC, and we identified a cell signaling pathway-based gene signature that may contribute to the development of combinatorial targeted therapies for breast cancer