Guanine quadruplexes (G4s) are an important structure of nucleic acids (DNA and RNA) with roles in several cellular processes. RNA G4s require specialized unwinding enzymes, of which only two have been previously identified. We describe the results of a simple and specific mass spectrometry guided method used to screen HEK293T cell lysate for G4 binding proteins. From these results, we validated the RNA helicase protein DDX21. DDX21 is an established RNA helicase, but has not yet been validated as a G4 binding protein. Through biochemical techniques, we confirm that DDX21-quadruplex RNA interactions are direct and mediated via a site of interaction at the C-terminus of the protein. Furthermore, through monitoring changes in nuclease sensitivity we show that DDX21 can unwind RNA G4. Finally, as proof of principle, we demonstrate the ability of DDX21 to suppress the expression of a protein with G4s in the 3΄ UTR of its mRNA.

The identification of four-stranded G-quadruplexes (G4s) has highlighted the fact that DNA has additional spatial organisations at its disposal other than double-stranded helices. Recently, it became clear that the formation of G4s is not limited to the traditional G3+NL1G3+NL2G3+NL3G3+ sequence motif. Instead, the G3 triplets can be interrupted by deoxythymidylate (DNA) or uridylate (RNA) where the base forms a bulge that loops out from the G-quadruplex core. Here, we report the first high-resolution X-ray structure of a unique unimolecular DNA G4 with a cytosine bulge. The G4 forms a dimer that is stacked via its 5′-tetrads. Analytical ultracentrifugation, static light scattering and small angle X-ray scattering confirmed that the G4 adapts a predominantly dimeric structure in solution. We provide a comprehensive comparison of previously published G4 structures containing bulges and report a special γ torsion angle range preferentially populated by the G4 core guanylates adjacent to bulges. Since the penalty for introducing bulges appears to be negligible, it should be possible to functionalize G4s by introducing artificial or modified nucleotides at such positions. The presence of the bulge alters the surface of the DNA, providing an opportunity to develop drugs that can specifically target individual G4s.

The environmental organism Serratia marcescens is one of the primary causes of numerous nosocomial outbreaks and opportunistic infections. Multi-drug resistance is now a common feature among S. marcescens clinical isolates, complicating the efficacy of treatment. Recent reports have attributed antibiotic resistance to altered porin expression as well as perturbation of the intrinsic AmpC beta-lactamase production pathway. In this study, we aimed to genetically correlate the absence of OmpF and OmpC classical porins with increased antibiotic resistance. In generating isogenic porin mutant strains, we avoided incorporating additional resistance through the use of antibiotic cassettes in gene replacement and adopted an alternative strategy in creating clean unmarked mutant strains. We found that lack of OmpF, but not OmpC, significantly increased antibiotic MIC values to the beta-lactam drugs such as ampicillin and cefoxitin as well as to nitrofurantoin. Furthermore, we found that cefoxitin did not induce intrinsic AmpC beta-lactamase production, indicating that the increased MIC values were a result of reduced permeability of cefoxitin due to the lack of OmpF. Genetic deletion of both ompF and ompC did not compromise the integrity of the bacterial cell envelope in optimal growth conditions, suggesting that other outer-membrane porins may function in a compensatory role to facilitate nutrient uptake and cell envelope integrity. Taken together, to our knowledge this is the first study that genetically correlates increased antibiotic resistance with altered porin expression in S. marcescens.

The production of recombinant proteins for functional and biophysical studies, especially in the field of structural determination, still represents a challenge as high quality and quantities are needed to adequately perform experiments. This is in part solved by optimizing protein constructs and expression conditions to maximize the yields in regular flask expression systems. Still, work flow and effort can be substantial with no guarantee to obtain improvements. This study presents a combination of workflows that can be used to dramatically increase protein production and improve processing results, specifically for the extracellular matrix protein Netrin-1. This proteoglycan is an axon guidance cue which interacts with various receptors to initiate downstream signaling cascades affecting cell differentiation, proliferation, metabolism, and survival. We were able to produce large glycoprotein quantities in mammalian cells, which were engineered for protein overexpression and secretion into the media using the controlled environment provided by a hollow fiber bioreactor. Close monitoring of the internal bioreactor conditions allowed for stable production over an extended period of time. In addition to this, Netrin-1 concentrations were monitored in expression media through biolayer interferometry which allowed us to increase Netrin-1 media concentrations tenfold over our current flask systems while preserving excellent protein quality and in solution behavior. Our particular combination of genetic engineering, cell culture system, protein purification, and biophysical characterization permitted us to establish an efficient and continuous production of high-quality protein suitable for structural biology studies that can be translated to various biological systems. Key points • Hollow fiber bioreactor produces substantial yields of homogenous Netrin-1 • Biolayer interferometry allows target protein quantitation in expression media • High production yields in the bioreactor do not impair Netrin-1 proteoglycan quality Graphical abstract

Boron neutron capture therapy (BNCT) is a two-step therapeutic process that utilizes Boron-10 in combination with low energy neutrons to effectively eliminate targeted cells. This therapy is primarily used for difficult to treat head and neck carcinomas; recent advances have expanded this method to cover a broader range of carcinomas. However, it still remains an unconventional therapy where one of the barriers for widespread adoption is the adequate delivery of Boron-10 to target cells. In an effort to address this issue, we examined a unique nanoparticle drug delivery system based on a highly stable and modular proteinaceous nanotube. Initially, we confirmed and structurally analyzed ortho-carborane binding into the cavities of the nanotube. The high ratio of Boron to proteinaceous mass and excellent thermal stability suggest the nanotube system as a suitable candidate for drug delivery into cancer cells. The full physicochemical characterization of the nanotube then allowed for further mechanistic molecular dynamic studies of the ortho-carborane uptake and calculations of corresponding energy profiles. Visualization of the binding event highlighted the protein dynamics and the importance of the interhelical channel formation to allow movement of the boron cluster into the nanotube. Additionally, cell assays showed that the nanotube can penetrate outer membranes of cancer cells followed by localization around the cells’ nuclei. This work uses an integrative approach combining experimental data from structural, molecular dynamics simulations and biological experiments to thoroughly present an alternative drug delivery device for BNCT which offers additional benefits over current delivery methods.

Netrin-1 is a bifunctional chemotropic guidance cue that plays key roles in diverse cellular processes including axon pathfinding, cell migration, adhesion, differentiation, and survival. Here, we present a molecular understanding of netrin-1 mediated interactions with glycosaminoglycan chains of diverse heparan sulfate proteoglycans (HSPGs) and short heparin oligosaccharides. Whereas interactions with HSPGs act as platform to co-localise netrin-1 close to the cell surface, heparin oligosaccharides have a significant impact on the highly dynamic behaviour of netrin-1. Remarkably, the monomer-dimer equilibrium of netrin-1 in solution is abolished in the presence of heparin oligosaccharides and replaced with highly hierarchical and distinct super assemblies leading to unique, yet unknown netrin-1 filament formation. In our integrated approach we provide a molecular mechanism for the filament assembly which opens fresh paths towards a molecular understanding of netrin-1 functions.

Netrin‐1 is well‐known for its chemoattractive and chemorepulsive properties for axon guidance. Early studies report that netrin‐1 inhibits granulocyte migration. On the other hand, netrin‐1 can promote cancer cell migration and invasion. The underlying mechanisms are not well understood, which requires more in‐depth characterizations of netrin‐1 mediated immune and cancer cell migration. The present study, for the first time, employs microfluidic devices that are recently developed to quantitatively investigate the effects of netrin‐1 on the motility and chemotaxis of human blood neutrophils and human breast cancer cells under well‐controlled gradient conditions. The results show that netrin‐1 reduces chemokinetic motility of human neutrophils, which is accompanied with reduced cell polarization and spreading. In addition, netrin‐1 reduces neutrophil chemotaxis to N‐formyl‐Met‐Leu‐Phe on fibronectin substrate but interestingly not on collagen substrate. By contrast, netrin‐1 promotes the migration of human breast cancer cells. Furthermore, it is found that netrin‐1 reduces neutrophil chemotaxis to the supernatant of human breast cancer cell culture. Collectively, this microfluidic cell migration study provides quantitative characterizations of the effects of netrin‐1 on the motility and chemotaxis of neutrophils and breast cancer cells, and further suggests the potential role of netrin‐1 in regulating neutrophil recruitment to breast cancer microenvironments.

We developed a single domain VHH multi-specific antibody format. Multispecific antibodies have multiple mechanisms of action which may work independently or together, to achieve better clinical outcomes in cancers with high unmet medical need such as SCLC. Here we describe the preclinical development of a trispecific antibody (KB-436) that targets Dopamine Receptor 2 (DRD2), PD-1 and CD47. DRD2 is a G protein-coupled receptor upregulated in many cancer types where it correlates with decreased patient survival. In pre-clinical studies DRD2 is associated with cancer cell stemness and tumor growth. Clinical responses were achieved with small molecules targeting DRD2 and dopaminergic drugs. In SCLC, representing 15% of lung cancers, 60-70% of patients showed high expression of DRD2. Checkpoint inhibition has shown some efficacy in lung cancer where PD-L1 inhibitors were approved as first line therapy in SCLC . SCLC patients rapidly fail chemotherapy, develop resistance to treatment including to immunotherapy associated with lack of tumor infiltrating immune cells, appearance of metastases and large numbers of circulating tumor cells. These observations suggest a link between DRD2 expression and resistance to treatment, making this receptor an attractive target for a multispecific therapy. CD47 is overexpressed by many cancers and is also expressed on lymphocytes. Here, targeting CD47 is integral to T-cell engagement and redirection, in a mechanism distinct from CD3 mediated T-cell engagement. The VHH modules of KB-436 (anti-DRD2, anti-PD-1 and anti-CD47) mediate multiple mechanisms of action to achieve anti-tumor effect. The anti-DRD2 VHH induces intracellular signaling, the anti-PD-1 VHH restores T cell function, and the anti-CD47 VHH recruits T cells without their generalized activation and blocks interaction of CD47 with SIRPa. Treatment with anti-DRD2 antibody significantly suppressed tumor growth in the DRD2-positive NCI-H510A SCLC model in SCID mice. KB-436 anti-tumor efficacy was tested in several in vivo immuno-oncology xenograft models of human SCLC and other solid tumors , reconstituted with human PBMC or with CD34+ hemopoietic stem cells. Treatment suppressed tumor growth, enhanced the in vivo effect of cisplatin-treatment in a less chemosensitive NCI-H69 variant, blocked metastases formation in CD34+ humanized NCG mice bearing established NCI-H69 tumors, and blocked metastases formation and increased survival in tail vein metastatic models. Trispecific KB-436 has a half-life in mice of around 5 days similar to that of other antibodies. It is produced at high yield (6 g/L) in a manufacturing cell line, conventional purification yields 99% purity and notably displays high stability under accelerated stability testing. In conclusion, the trispecific KB-436 antibody, has strong in vivo anti-tumor activity mediated via multiple mechanisms of action, is easily expressed and purified and is very stable. Together, this data supports the clinical development of KB-436 in advanced metastatic solid cancer indications, including SCLC. Citation Format: Shugang Yao, Yun Cui, Anna Kazanats, Liying Gong, Claire Bonfils, Dominic Hou, Emily Chen, Elijus Undzys, Jacynthe Toulouse, Milica Krstic, Hiba Zahreddine, Israel Matos, Alex Zhou, Aniel Moya-Torres, Richard Wargachuk, Carl Gay, Lauren Byers, Gordon Ngan, Luis da Cruz, David Young. Pre-clinical development of a dopamine receptor 2, PD-1 and CD47 trispecific antibody for treatment of small cell lung cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P200.