Structures of Cryptococcus neoformans protein farnesyltransferase reveal strategies for developing inhibitors that target fungal pathogens.

Cryptococcus neoformans is a fungal pathogen that causes life-threatening infections in immunocompromised individuals, including AIDS patients and transplant recipients. Few antifungals can treat C. neoformans infections, and drug resistance is increasing. Protein farnesyltransferase (FTase) catalyzes post-translational lipidation of key signal transduction proteins and is essential in C. neoformans. We present a multidisciplinary study validating C. neoformans FTase (CnFTase) as a drug target, showing that several anticancer FTase inhibitors with disparate scaffolds can inhibit C. neoformans and suggesting structure-based strategies for further optimization of these leads. Structural studies are an essential element for species-specific inhibitor development strategies by revealing similarities and differences between pathogen and host orthologs that can be exploited. We, therefore, present eight crystal structures of CnFTase that define the enzymatic reaction cycle, basis of ligand selection, and structurally divergent regions of the active site. Crystal structures of clinically important anticancer FTase inhibitors in complex with CnFTase reveal opportunities for optimization of selectivity for the fungal enzyme by modifying functional groups that interact with structurally diverse regions. A substrate-induced conformational change in CnFTase is observed as part of the reaction cycle, a feature that is mechanistically distinct from human FTase. Our combined structural and functional studies provide a framework for developing FTase inhibitors to treat invasive fungal infections.

RF1 attenuation enables efficient non-natural amino acid incorporation for production of homogeneous antibody drug conjugates.

Amber codon suppression for the insertion of non-natural amino acids (nnAAs) is limited by competition with release factor 1 (RF1). Here we describe the genome engineering of a RF1 mutant strain that enhances suppression efficiency during cell-free protein synthesis, without significantly impacting cell growth during biomass production. Specifically, an out membrane protease (OmpT) cleavage site was engineered into the switch loop of RF1, which enables its conditional inactivation during cell lysis. This facilitates extract production without additional processing steps, resulting in a scaleable extract production process. The RF1 mutant extract allows nnAA incorporation at previously intractable sites of an IgG1 and at multiple sites in the same polypeptide chain. Conjugation of cytotoxic agents to these nnAAs, yields homogeneous antibody drug conjugates (ADCs) that can be optimized for conjugation site, drug to antibody ratio (DAR) and linker-warheads designed for efficient tumor killing. This platform provides the means to generate therapeutic ADCs inaccessible by other methods that are efficient in their cytotoxin delivery to tumor with reduced dose-limiting toxicities and thus have the potential for better clinical impact.