RESUMO
Therapeutic monoclonal antibodies have transformed medicine, especially with regards to treating cancers and disorders of the immune system. More than 50 antibody-derived drugs have already reached the clinic, the majority of which target cytokines or cell-surface receptors. Unfortunately, many of these targets have pleiotropic functions: they serve multiple different roles, and often not all of these roles are disease-related. This can be problematic because antibodies act throughout the body, and systemic neutralization of such targets can lead to safety concerns. To address this, we have developed a strategy whereby an antibody's ability to recognize its antigen is modulated by a second layer of control, relying on addition of an exogenous small molecule. In previous studies, we began to explore this idea by introducing a deactivating tryptophan-to-glycine mutation in the domain-domain interface of a single-chain variable fragment (scFv), and then restoring activity by adding back indole to fit the designed cavity. Here, we now describe a novel computational strategy for enumerating larger cavities that can be formed by simultaneously introducing multiple adjacent large-to-small mutations; we then carry out a complementary virtual screen to identify druglike compounds to match each candidate cavity. We first demonstrate the utility of this strategy in a fluorescein-binding single-chain variable fragment (scFv) and experimentally characterize a triple mutant with reduced antigen-binding (Rip-3) that can be rescued using a complementary ligand (Stitch-3). Because our design is built upon conserved residues in the antibody framework, we then show that the same mutation/ligand pair can also be used to modulate antigen-binding in an scFv build from a completely unrelated framework. This set of residues is present in many therapeutic antibodies as well, suggesting that this mutation/ligand pair may serve as a general starting point for introducing ligand-dependence into many clinically relevant antibodies.
RESUMO
Promysalin is a small-molecule natural product that specifically inhibits growth of the Gram-negative pathogen Pseudomonas aeruginosa (PA). This activity holds promise in the treatment of multidrug resistant infections found in immunocompromised patients with chronic illnesses, such as cystic fibrosis. In 2015, our lab completed the first total synthesis; subsequent analogue design and SAR investigation enabled identification of succinate dehydrogenase (Sdh) as the biological target in PA. Herein, we report the target-guided design of new promysalin analogues with varying alkyl chains, one of which is on par with our most potent analogue to date. Computational docking revealed that some analogues have a different orientation in the Sdh binding pocket, placing the terminal carbon proximal to a tryptophan residue. This inspired the design of an extended side chain analogue bearing a terminal phenyl moiety, providing a basis for the design of future analogues.
Assuntos
Salicilamidas , Succinato Desidrogenase , Humanos , Pirrolidinas , Succinato Desidrogenase/metabolismoRESUMO
Cyclin-dependent kinase 9 (CDK9) promotes transcriptional elongation through RNAPII pause release. We now report that CDK9 is also essential for maintaining gene silencing at heterochromatic loci. Through a live cell drug screen with genetic confirmation, we discovered that CDK9 inhibition reactivates epigenetically silenced genes in cancer, leading to restored tumor suppressor gene expression, cell differentiation, and activation of endogenous retrovirus genes. CDK9 inhibition dephosphorylates the SWI/SNF protein BRG1, which contributes to gene reactivation. By optimization through gene expression, we developed a highly selective CDK9 inhibitor (MC180295, IC50 = 5 nM) that has broad anti-cancer activity in vitro and is effective in in vivo cancer models. Additionally, CDK9 inhibition sensitizes to the immune checkpoint inhibitor α-PD-1 in vivo, making it an excellent target for epigenetic therapy of cancer.
Assuntos
Quinase 9 Dependente de Ciclina/metabolismo , Animais , Linhagem Celular Tumoral , Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Quinase 9 Dependente de Ciclina/genética , DNA Helicases/genética , DNA Helicases/metabolismo , Metilação de DNA , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-Atividade , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Monoclonal antibody therapeutics have revolutionized the treatment of diseases such as cancer and autoimmune disorders, and also serve as research reagents for diverse and unparalleled applications. To extend their utility in both contexts, we have begun development of tunable antibodies, whose activity can be controlled by addition of a small molecule. Conceptually, we envision that incorporating cavity-forming mutations into an antibody can disrupt its structure, thereby reducing its affinity for antigen; addition of a small molecule may then restore the active structure, and thus rescue antigen binding. As a first proof of concept toward implementing this strategy, we have incorporated individual tryptophan to glycine mutations into FITC-E2, an anti-fluorescein single-chain variable fragment (scFv). We find that these can disrupt the protein structure and diminish antigen binding, and further that both structure and function can be rescued by addition of indole to complement the deleted side chain. While the magnitude of the affinity difference triggered by indole is modest in this first model system, it nonetheless provides a framework for future mutation/ligand pairs that may induce more dramatic responses. Disrupting and subsequently rescuing antibody activity, as exemplified by this first example, may represent a new approach to "design in" fine-tuned control of antibody activity for a variety of future applications.
Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Engenharia de Proteínas/métodos , Substituição de Aminoácidos , Anticorpos Monoclonais/genética , Fluoresceína-5-Isotiocianato/química , Fluoresceína-5-Isotiocianato/metabolismo , Fluorescência , Glicina/genética , Indóis/química , Modelos Moleculares , Mutagênese Sítio-Dirigida/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/metabolismo , Relação Estrutura-Atividade , Triptofano/genéticaRESUMO
Protein-protein interactions represent an exciting and challenging target class for therapeutic intervention using small molecules. Protein interaction sites are often devoid of the deep surface pockets presented by "traditional" drug targets, and crystal structures reveal that inhibitors typically engage these sites using very shallow binding modes. As a consequence, modern virtual screening tools developed to identify inhibitors of traditional drug targets do not perform as well when they are instead deployed at protein interaction sites. To address the need for novel inhibitors of important protein interactions, here we introduce an alternate docking strategy specifically designed for this regime. Our method, termed DARC (Docking Approach using Ray-Casting), matches the topography of a surface pocket "observed" from within the protein to the topography "observed" when viewing a potential ligand from the same vantage point. We applied DARC to carry out a virtual screen against the protein interaction site of human antiapoptotic protein Mcl-1 and found that four of the top-scoring 21 compounds showed clear inhibition in a biochemical assay. The Ki values for these compounds ranged from 1.2 to 21 µM, and each had ligand efficiency comparable to promising small-molecule inhibitors of other protein-protein interactions. These hit compounds do not resemble the natural (protein) binding partner of Mcl-1, nor do they resemble any known inhibitors of Mcl-1. Our results thus demonstrate the utility of DARC for identifying novel inhibitors of protein-protein interactions.