Engineered living materials have the potential for wide-ranging applications such as biosensing and treatment of diseases. Programmable cells provide the functional basis for living materials; however, their release into the environment raises numerous biosafety concerns. Current designs that limit the release of genetically engineered cells typically involve the fabrication of multilayer hybrid materials with submicrometer porous matrices. Nevertheless the stringent physical barriers limit the diffusion of macromolecules and therefore the repertoire of molecules available for actuation in response to communication signals between cells and their environment. Here, we engineer a novel living material entitled “Platform for Adhesin-mediated Trapping of Cells in Hydrogels” (PATCH). This technology is based on engineered E. coli that displays an adhesion protein derived from an Antarctic bacterium with a high affinity for glucose. The adhesin stably anchors E. coli in dextran-based hydrogels with large pore diameters (10–100 μm) and reduces the leakage of bacteria into the environment by up to 100-fold. As an application of PATCH, we engineered E. coli to secrete the bacteriocin lysostaphin which specifically kills Staphyloccocus aureus with low probability of raising antibiotic resistance. We demonstrated that living materials containing this lysostaphin-secreting E. coli inhibit the growth of S. aureus, including the strain resistant to methicillin (MRSA). Our tunable platform allows stable integration of programmable cells in dextran-based hydrogels without compromising free diffusion of macromolecules and could have potential applications in biotechnology and biomedicine.

The RORγt nuclear receptor (NR) is of critical importance for the differentiation and proliferation of T helper 17 (Th17) cells and their production of the pro-inflammatory cytokine IL-17a. Dysregulation of RORγt has been linked to various autoimmune diseases, and small molecule inhibition of RORγt is therefore an attractive strategy to treat these diseases. RORγt is a unique NR in that it contains both a canonical, orthosteric and a second, allosteric ligand binding site in its ligand binding domain (LBD). Hence, dual targeting of both binding pockets constitutes an attractive alternative molecular entry for pharmacological modulation. Here, we report a chemical biology approach to develop a bitopic ligand for the RORγt NR, enabling concomitant engagement of both binding pockets. Three candidate bitopic ligands, Bit-L15 , Bit-L9 , and Bit-L4 , comprising an orthosteric and allosteric RORγt pharmacophore linked via a polyethylene glycol (PEG) linker, were designed, synthesized, and evaluated to examine the influence of linker length on the RORγt binding mode. Bit-L15 and Bit-L9 show convincing evidence of concomitant engagement of both RORγt binding pockets, while the shorter Bit-L4 does not show this evidence, as was anticipated during the ligand design. As the most potent bitopic RORγt ligand, Bit-L15 , antagonizes RORγt function in a potent manner in both a biochemical and cellular context. Furthermore, Bit-L15 displays an increased selectivity for RORγt over RORα and PPARγ compared to the purely orthosteric and allosteric parent compounds. Combined, these results highlight potential advantages of bitopic NR modulation over monovalent targeting strategies.

The inhibition of the nuclear receptor retinoic-acid-receptor-related orphan receptor γt (RORγt) is a promising strategy in the treatment of autoimmune diseases. RORγt features an allosteric binding site within its ligand-binding domain that provides an opportunity to overcome drawbacks associated with orthosteric modulators. Recently, trisubstituted isoxazoles were identified as a novel class of allosteric RORγt inverse agonists. This chemotype offers new opportunities for optimization into selective and efficacious allosteric drug-like molecules. Here, we explore the structure–activity relationship profile of the isoxazole series utilizing a combination of structure-based design, X-ray crystallography, and biochemical assays. The initial lead isoxazole ( FM26 ) was optimized, resulting in compounds with a ∼10-fold increase in potency (low nM), significant cellular activity, promising pharmacokinetic properties, and a good selectivity profile over the peroxisome-proliferated-activated receptor γ and the farnesoid X receptor. We envisage that this work will serve as a platform for the accelerated development of isoxazoles and other novel chemotypes for the effective allosteric targeting of RORγt.

5 Stichting PAMM, Laboratory for pathology and medical microbiology, De Run 6250, 5504 DL Veldhoven 6 Laboratory of Physical Chemistry 7 Molecular biosensing for medical diagnostics 8 Laboratory of protein engineering 1-4, 6-8 : Abstract:Engineered living materials have the potential for wide-ranging applications such as biosensing and treatment of diseases. Programmable cells provide the functional basis for living materials, however, their release into the environment raises numerous biosafety concerns. Current designs that limit the release of genetically engineered cells typically involve the fabrication of multi-layer hybrid materials with sub-micron porous matrices. Nevertheless the stringent physical barriers limit the diffusion of macromolecules and therefore the repertoire of molecules available for actuation in response to communication signals between cells and their environment. Here, we engineer a first-of-its-kind living material entitled 'Platform for Adhesin-mediated Trapping of Cells in Hydrogels' (PATCH). This technology is based on engineered E. coli that displays an adhesion protein derived from an Antarctic bacterium with high affinity for glucose. The adhesin stably anchors E. coli in dextran-based hydrogels with large pore diameters (10-100 µm) and reduces the leakage of bacteria into the environment by up to 100-fold. As an application of PATCH, we engineered E. coli to secrete lysostaphin via the Type 1 Secretion System and demonstrated that living materials containing this E. coli inhibit the growth of S. aureus, including the strain resistant to methicillin (MRSA). Our tunable platform allows stable integration of programmable cells in dextran-based hydrogels without compromising free diffusion of macromolecules and could have potential applications in biotechnology and biomedicine. Introduction:Synthetic biology aims to design programmable cells that combine sensing and molecular computing operations with on-demand production of proteins that have a broad spectrum of therapeutic applications [1][2][3][4]. Engineered living materials (ELMs) integrate genetically engineered cells with free standing materials and represent a new class of environmentally responsive living devices with designer physicochemical and material properties [5][6][7]. Ideally, ELMs provide mechanical robustness to engineered cells, prevent their leakage to the environment and allow cells to be viable for extended periods of time. The containment of genetically-modified microorganisms (GMMs) within various materials has become a grand challenge for future synthetic biology applications [8]. To date, strategies for containing GMMs inside a living device are based on the physical confinement by multi-layer materials [9][10][11]. Hybrid micro-patterned devices combining layers of elastomer and microporous hydrogel enabled the exchange of information with surrounding environment via diffusion of chemical inducers and their sensing by GMMs while displaying high mechanical resilience [10]. Nevertheless, the low porosity ...