Exploring rigid-backbone protein docking in biologics discovery: a test using the DARPin scaffold

Accurate protein-protein docking remains challenging, especially for artificial biologics not coevolved naturally against their protein targets, like antibodies and other engineered scaffolds. We previously developed ProPOSE, an exhaustive docker with full atomistic details, which delivers cutting-edge performance by allowing side-chain rearrangements upon docking. However, extensive protein backbone flexibility limits its practical applicability as indicated by unbound docking tests. To explore the usefulness of ProPOSE on systems with limited backbone flexibility, here we tested the engineered scaffold DARPin, which is characterized by its relatively rigid protein backbone. A prospective screening campaign was undertaken, in which sequence-diversified DARPins were docked and ranked against a directed epitope on the target protein BCL-W. In this proof-of-concept study, only a relatively small set of 2,213 diverse DARPin interfaces were selected for docking from the huge theoretical library from mutating 18 amino-acid positions. A computational selection protocol was then applied for enrichment of binders based on normalized computed binding scores and frequency of binding modes against the predefined epitope. The top-ranked 18 designed DARPin interfaces were selected for experimental validation. Three designs exhibited binding affinities to BCL-W in the nanomolar range comparable to control interfaces adopted from known DARPin binders. This result is encouraging for future screening and engineering campaigns of DARPins and possibly other similarly rigid scaffolds against targeted protein epitopes. Method limitations are discussed and directions for future refinements are proposed

The Prevalence, Management and Impact of Dysmenorrhea on Medical Students’ Lives—A Multicenter Study

Introduction: Dysmenorrhea is defined as the presence of painful menstruation, and it affects daily activities in different ways. The aims of this study were to assess the prevalence and management of dysmenorrhea and to determine the impact of dysmenorrhea on the quality of life of medical students. Material and methods: The study conducted was prospective, analytical and observational and was performed between 7 November 2019 and 30 January 2020 in five university centers from Romania. The data was collected using an original questionnaire regarding menstrual cycles and dysmenorrhea. The information about relationships with family or friends, couples’ relationships and university activity helped to assess the effects of dysmenorrhea on quality of life. The level of significance was set at p < 0.05. Results: The study comprised 1720 students in total. The prevalence of dysmenorrhea was 78.4%. During their menstrual period, most female students felt more agitated or nervous (72.7%), more tired (66.9%), as if they had less energy for daily activities (75.9%) and highly stressed (57.9%), with a normal diet being difficult to achieve (30.0%). University courses (49.4%), social life (34.5%), couples’ relationships (29.6%), as well as relationships with family (21.4%) and friends (15.4%) were also affected, depending on the duration and intensity of the pain. Conclusion: Dysmenorrhea has a high prevalence among medical students and could affect the quality of life of students in several ways. During their menstrual period, most female students feel as if they have less energy for daily activities and exhibit a higher level of stress. The intensity of the symptoms varies considerably and, with it, the degree of discomfort it creates. Most student use both pharmacological and non-pharmacological methods to reduce pain (75.7%). University courses, social life, couples’ relationships, as well as relationships with family and friends are affected, depending on the duration and intensity of the pain

Solvated interaction energy: from small-molecule to antibody drug design

Scoring functions are ubiquitous in structure-based drug design as an aid to predicting binding modes and estimating binding affinities. Ideally, a scoring function should be broadly applicable, obviating the need to recalibrate and refit its parameters for every new target and class of ligands. Traditionally, drugs have been small molecules, but in recent years biologics, particularly antibodies, have become an increasingly important if not dominant class of therapeutics. This makes the goal of having a transferable scoring function, i.e., one that spans the range of small-molecule to protein ligands, even more challenging. One such broadly applicable scoring function is the Solvated Interaction Energy (SIE), which has been developed and applied in our lab for the last 15 years, leading to several important applications. This physics-based method arose from efforts to understand the physics governing binding events, with particular care given to the role played by solvation. SIE has been used by us and many independent labs worldwide for virtual screening and discovery of novel small-molecule binders or optimization of known drugs. Moreover, without any retraining, it is found to be transferrable to predictions of antibody-antigen relative binding affinities and as accurate as functions trained on protein-protein binding affinities. SIE has been incorporated in conjunction with other scoring functions into ADAPT (Assisted Design of Antibody and Protein Therapeutics), our platform for affinity modulation of antibodies. Application of ADAPT resulted in the optimization of several antibodies with 10-to-100-fold improvements in binding affinity. Further applications included broadening the specificity of a single-domain antibody to be cross-reactive with virus variants of both SARS-CoV-1 and SARS-CoV-2, and the design of safer antibodies by engineering of a pH switch to make them more selective towards acidic tumors while sparing normal tissues at physiological pH

Programmable Attenuation of Antigenic Sensitivity for a Nanobody-Based EGFR Chimeric Antigen Receptor Through Hinge Domain Truncation

Epidermal growth factor family receptor (EGFR) is commonly overexpressed in many solid tumors and an attractive target for chimeric antigen receptor (CAR)-T therapy, but as EGFR is also expressed at lower levels in healthy tissues a therapeutic strategy must balance antigenic responsiveness against the risk of on-target off-tumor toxicity. Herein, we identify several camelid single-domain antibodies (also known as nanobodies) that are effective EGFR targeting moieties for CARs (EGFR-sdCARs) with very strong reactivity to EGFR-high and EGFR-low target cells. As a strategy to attenuate their potent antigenic sensitivity, we performed progressive truncation of the human CD8 hinge commonly used as a spacer domain in many CAR constructs. Single amino acid hinge-domain truncation progressively decreased both EGFR-sdCAR-Jurkat cell binding to EGFR-expressing targets and expression of the CD69 activation marker. Attenuated signaling in hinge-truncated EGFR-sdCAR constructs increased selectivity for antigen-dense EGFR-overexpressing cells over an EGFR-low tumor cell line or healthy donor derived EGFR-positive fibroblasts. We also provide evidence that epitope location is critical for determining hinge-domain requirement for CARs, as hinge truncation similarly decreased antigenic sensitivity of a membrane-proximal epitope targeting HER2-CAR but not a membrane-distal EGFRvIII-specific CAR. Hinge-modified EGFR-sdCAR cells showed clear functional attenuation in Jurkat-CAR-T cells and primary human CAR-T cells from multiple donors in vitro and in vivo. Overall, these results indicate that hinge length tuning provides a programmable strategy for throttling antigenic sensitivity in CARs targeting membrane-proximal epitopes, and could be employed for CAR-optimization and improved tumor selectivity

Nature versus nurture in two highly enantioselective esterases from Bacillus cereus and Thermoanaerobacter tengcongensis

There is an increasing need for the use of biocatalysis to obtain enantiopure compounds as chiral building blocks for drug synthesis such as antibiotics. The principal findings of this study are: i) the complete sequenced genomes of Bacillus cereus ATCC 14579 and Thermoanaerobacter tengcongensis MB4 contain a hitherto undescribed enantioselective and alkaliphilic esterase (BcEST and TtEST, respectively) that is specific for the production of (R)-2-benzyloxy-propionic acid ethyl ester, a key intermediate in the synthesis of levofloxacin, a potent antibiotic; and, ii) directed evolution targeted for increased thermostability of BcEST produced two improved variants, but in either case the 3 \u2013 5\ub0C increase in the apparent melting temperature (Tm) of the mutants over the native BcEST that has a Tm of 50\ub0C was outperformed by TtEST, a naturally-occurring homolog with a Tm of 65\ub0C. Protein modeling of BcEST mapped the S148C and K272R mutations at protein surface and the I88T and Q110L mutations at more buried locations. This work expands the repertoire of characterized members of the \u3b1/\u3b2 fold-hydrolase superfamily. Further, it shows that genome mining is an economical option for new biocatalyst discovery and we provide a rare example of a naturally-occurring thermostable biocatalyst that outperforms experimentally-evolved homologs that carry out the same hydrolysis.Peer reviewed: YesNRC publication: Ye

A rational engineering strategy for designing protein a-binding camelid single-domain antibodies

Staphylococcal protein A (SpA) and streptococcal protein G (SpG) affinity chromatography are the gold standards for purifying monoclonal antibodies (mAbs) in therapeutic applications. However, camelid VHH single-domain Abs (sdAbs or VHHs) are not bound by SpG and only sporadically bound by SpA. Currently, VHHs require affinity tag-based purification, which limits their therapeutic potential and adds considerable complexity and cost to their production. Here we describe a simple and rapid mutagenesis-based approach designed to confer SpA binding upon a priori non-SpA-binding VHHs. We show that SpA binding of VHHs is determined primarily by the same set of residues as in human mAbs, albeit with an unexpected degree of tolerance to substitutions at certain core and non-core positions and some limited dependence on at least one residue outside the SpA interface, and that SpA binding could be successfully introduced into five VHHs against three different targets with no adverse effects on expression yield or antigen binding. Next-generation sequencing of llama, alpaca and dromedary VHH repertoires suggested that species differences in SpA binding may result from frequency variation in specific deleterious polymorphisms, especially Ile57. Thus, the SpA binding phenotype of camelid VHHs can be easily modulated to take advantage of tag-less purification techniques, although the frequency with which this is required may depend on the source species

“Trait EI in the relationship between needs fulfilment and symptoms and attitudes associated with EDs”

Eating disorders (EDs) are a set of pathologies, which have been increasing in prevalence in the recent years, suggesting the importance of studying symptoms and attitudes associated with EDs in depth. Several studies have showed that both psychological basic needs and trait emotion intelligence (trait EI) are relevant aspects of EDs, however these two aspects were never tested concurrently. Previous studies have shown that self-determined motivation could be a plausible antecedent that may account for individual variation in trait EI, and for this reason, it seems to be extremely relevant to integrate trait EI in a Self-Determination Theory (SDT) framework. The aim of this study is to test a mediation model of trait EI in the relationship between need fulfilment and eating disorders. In a sample of 159 females aged between 16 and 22 years old (M = 18.71; SD = 1.98) instruments were administered to measure the basic psychological needs, trait EI, and eating disorders. Results of this study showed that need fulfilment was negatively related to eating disorders and positively related to trait EI, whereas trait EI was negatively related to eating disorders. Furthermore, trait EI has shown a mediation role in the relation between basic psychological needs and eating disorders