Synthesis and Optimization of 1-Substituted Imidazo[4,5-c]quinoline TLR7 Agonists.

TLR7 agonists have significant therapeutic potential in a variety of oncology and autoimmune applications. We recently reported a potent TLR7 selective agonist 1 that could be delivered by antibody-drug conjugate (ADC) technology to elicit potent anticancer activity. Herein we report synthetic chemistry and structure-activity relationship studies to develop TLR7 agonists with improved potency for next-generation ADC efforts. We found that the addition of hydrophobic acyl tails to parent compound 1 generally resulted in retained or improved TLR7 agonist activity without sacrificing the permeability or the selectivity over TLR8. In contrast, the addition of a simple alkyl tail at the same position resulted in a dramatic loss in potency. Molecular modeling was performed to provide a rationale for this dramatic loss in potency. We ultimately identified compounds 17b, 16b, and 16d as highly potent TLR7 agonists that potently induced the activation of mouse macrophages and hPBMCs at low-nanomolar concentrations.

Rational Design of Self-Assembling Artificial Proteins Utilizing a Micelle-Assisted Protein Labeling Technology (MAPLabTech): Testing the Scope.

Self-assembling artificial proteins (SAPs) have gained enormous interest in recent years due to their applications in different fields. Synthesis of well-defined monodisperse SAPs is accomplished predominantly through genetic methods. However, the last decade has witnessed the use of a few chemical technologies for this purpose. In particular, micelle-assisted protein labeling technology (MAPLabTech) has made huge progress in this area. The first generation MAPLabTech focused on site-specific labeling of the active-site residue of serine proteases to make SAPs. Further, this methodology was exploited for labeling of N-terminal residue of a globular protein to make functional SAPs. In this study, we describe the synthesis of novel SAPs by developing a chemical method for site-specific labeling of a surface-exposed cysteine residue of globular proteins. In addition, we disclose the synthesis of redox-sensitive SAPs and their systematic self-assembly and disassembly studies using size-exclusion chromatography. Altogether these studies further expand the scope of MAPLabTech in different fields such as vaccine design, targeted drug delivery, diagnostic imaging, biomaterials, and tissue engineering.

A Universal Chemical Method for Rational Design of Protein-Based Nanoreactors*.

Self-assembly of a monomeric protease to form a multi-subunit protein complex "proteasome" enables targeted protein degradation in living cells. Naturally occurring proteasomes serve as an inspiration and blueprint for the design of artificial protein-based nanoreactors. Here we disclose a general chemical strategy for the design of proteasome-like nanoreactors. Micelle-assisted protein labeling (MAPLab) technology along with the N-terminal bioconjugation strategy is utilized for the synthesis of a well-defined monodisperse self-assembling semi-synthetic protease. The designed protein is programmed to self-assemble into a proteasome-like nanostructure which preserves the functional properties of native protease.

Rapid Chemical Synthesis of Self-Assembling Semi-Synthetic Proteins.

The design of well-defined monodispersed self-assembling semi-synthetic proteins is emerging as a promising research avenue. These proteins hold great potential to be used as scaffolds for various protein nanotechnology applications. Currently, there are very few chemical methods reported; however, they suffer from elaborate multistep organic synthesis. Herein, we report a new chemical methodology for the rapid synthesis of a diverse set of semi-synthetic protein families, which include protein amphiphiles, facially amphiphilic protein-dendron conjugates, and pH-sensitive protein-dendron conjugates. This chemical method holds great potential to access a wide variety of semi-synthetic proteins in a short time.

Design, Synthesis, and Self-Assembly Studies of a Suite of Monodisperse, Facially Amphiphilic, Protein-Dendron Conjugates.

The custom design of protein-dendron amphiphilic macromolecules is at the forefront of macromolecular engineering. Macromolecules with this architecture are very interesting because of their ability to self-assemble into various biomimetic nanoscopic structures. However, to date, there are no reports on this concept due to technical challenges associated with the chemical synthesis. Towards that end, herein, a new chemical methodology for the modular synthesis of a suite of monodisperse, facially amphiphilic, protein-dendron bioconjugates is reported. Benzyl ether dendrons of different generations (G1-G4) are coupled to monodisperse cetyl ethylene glycol to form macromolecular amphiphilic activity-based probes (AABPs) with a single protein reactive functionality. Micelle-assisted protein labeling technology is utilized for site-specific conjugation of macromolecular AABPs to globular proteins to make monodisperse, facially amphiphilic, protein-dendron bioconjugates. These biohybrid conjugates have the ability to self-assemble into supramolecular protein nanoassemblies. Self-assembly is primarily mediated by strong hydrophobic interactions of the benzyl ether dendron domain. The size, surface charge, and oligomeric state of protein nanoassemblies could be systematically tuned by choosing an appropriate dendron or protein of interest. This chemical method discloses a new way to custom-make monodisperse, facially amphiphilic, protein-dendron bioconjugates.

Rational Design of Supramolecular Dynamic Protein Assemblies by Using a Micelle-Assisted Activity-Based Protein-Labeling Technology.

The self-assembly of proteins into higher-order superstructures is ubiquitous in biological systems. Genetic methods comprising both computational and rational design strategies are emerging as powerful methods for the design of synthetic protein complexes with high accuracy and fidelity. Although useful, most of the reported protein complexes lack a dynamic behavior, which may limit their potential applications. On the contrary, protein engineering by using chemical strategies offers excellent possibilities for the design of protein complexes with stimuli-responsive functions and adaptive behavior. However, designs based on chemical strategies are not accurate and therefore, yield polydisperse samples that are difficult to characterize. Here, we describe simple design principles for the construction of protein complexes through a supramolecular chemical strategy. A micelle-assisted activity-based protein-labeling technology has been developed to synthesize libraries of facially amphiphilic synthetic proteins, which self-assemble to form protein complexes through hydrophobic interaction. The proposed methodology is amenable for the synthesis of protein complex libraries with molecular weights and dimensions comparable to naturally occurring protein cages. The designed protein complexes display a rich structural diversity, oligomeric states, sizes, and surface charges that can be engineered through the macromolecular design. The broad utility of this method is demonstrated by the design of most sophisticated stimuli-responsive systems that can be programmed to assemble/disassemble in a reversible/irreversible fashion by using the pH or light as trigger.