Chemical Biology Discussion Group Year-End Meeting

Abstracts

Computationally Guided Drug Discovery

Bill Jorgensen, PhD, Yale University

Drug development is being pursued through computer-aided structure-based design. For de novo lead generation, the BOMB program builds combinatorial libraries in a protein binding site using a selected core and substituents, and QikProp is applied to filter all designed molecules to ensure that they have drug-like properties. Monte Carlo/free-energy perturbation simulations are then executed to refine the predictions for the best scoring leads including ca. 1000 explicit water molecules and extensive sampling for the protein and ligand.

FEP calculations for optimization of substituents on an aromatic ring and for choice of heterocycles are now common. Alternatively, docking with Glide is performed with large databases of purchasable compounds to provide leads, which are then optimized via the FEP-guided route. Successful application has been achieved for HIV reverse transcriptase, FGFR1 kinase, and macrophage migration inhibitory factor (MIF); micromolar leads have been rapidly advanced to extraordinarily potent inhibitors.

Chemical Tailoring of Mammalian Viral Vectors via the Incorporation of Unnatural Sugars and Amino Acids

Partha Banerjee, Stony Brook University

Virothapy applications have been limited by the lack of general, efficient and non-perturbing methods to alter virus surface functionality. Genetic methods generally perturb virus physiology and lack access to many desirable effector molecules, whereas traditional chemical modifications lack the control of genetics. Here we demonstrate a two step labeling technique of adenoviral virus capsid proteins, an initial metabolic “prelabeling” with unnatural substrates during virus production that allows subsequent access to highly selective bioorthogonal reactions facilitating the attachment of a variety of effector functionality onto the coat proteins. The metabolic incorporation of the unnatural substrates demonstrates no significant impact on virus production or infectivity. This novel technique utilizes a non-natural sugar, N-azidoacetylgalactosamine and an amino acid moiety, azidohomoalanine to substitute N-acetylglucosamine and methionine residues respectively in the virus coat protein makeup. Copper catalyzed “Click” and Staudinger ligation reactions have been used to append a variety of probes to solvent exposed azides. Modification of the incorporated azide functionality with cancer selective targeting ligands demonstrates markedly increased gene delivery in breast cancer cell lines.

Effort Toward the Total Synthesis of Aconitine Norditerpenoid Alkaloid 9-Deoxylappaconitine

Yuan Shi, Memorial Sloan Kettering Cancer Center

Aconitine norditerpenoid alkaloids are a large family of natural products isolated from plant genera Delphinium and Aconitum. Many aconitine alkaloids are found to be potent sodium ion channel modulators. Synthetic studies toward aconitine alkaloids have been ongoing for decades, wherein successful total synthesis has only been accomplished by Wiesner and coworkers thirty years ago. We are in the process of developing a promising convergent strategy to this class of alkaloids. Thus, the synthesis of the complete aconitine skeleton was achieved via a sequence involving N-acyliminium cyclization and radical cyclization. These findings are currently being applied to the total synthesis of 9-deoxylappaconitine.

Analysis of Helix-Mediated Protein-Protein Interactions for Potential Therapeutic Intervention

Andrea Jochim, New York University

This talk will discuss the identity and analysis of helical protein interfaces as potential targets for synthetic modulators of protein–protein interactions. We were inspired in our undertaking by previous studies to determine the number and class of protein drug targets wherein it was determined that less than 400 druggable domains cover all current drug targets—a number that compares poorly with the projected number of protein families. We have assessed the available data on protein–protein complexes with helical interfaces from the Protein Data Bank. Our endeavor has a dual purpose: to provide a dataset for the chemical biology community representing the variety and number of targets available for helix mimetics, and to examine the nature of helices that appear in interface proteins.

A Heritable Mutagenesis System for Directed Evolution Entirely Within Living Cells

Dante Romanini, Columbia University

Directed evolution reigns as the most powerful technique for the generation of biomolecules with new properties and functions, yet its cyclical process of randomization, screening or selection, and amplification of the winning sequences remains labor- and resource-intensive. In contrast to the standard practice of DNA randomization in vitro followed by transformation of the library into a host cell for protein expression, we sought to create a fully in vivo mutagenesis system that would allow for the entire directed evolution cycle to take place within the cell. Specifically, we have developed a method that relies on homologous recombination to introduce cassette-encoded mutations into a gene of interest within yeast cells. The mutagenesis is inducible, proceeds with high efficiency, and is compatible with the sexual reproduction pathway of yeast, allowing desirable sequences to be exchanged among individual members of the cell population. In this way, the most beneficial mutations from very large random libraries can be combined entirely in vivo, without intermediate human manipulation of the coding DNA, to discover proteins with useful new functions.

Discovery of Antibacterial Virulence Inhibitors Targeted at Type III Protein Secretion

Lun K. Tsou, PhD, The Rockefeller University

Type III secretion systems (T3SSs) are used by Gram-negative bacterial pathogens to inject effector proteins into host cells allowing infection and intracellular replication. We developed a two-component enzymatic reporter system generated by fusing caboxy-peptidase G2 (CPG2) to SopE2 effector of Salmonella typhimurium and validated this fusion protein is efficiently secreted via T3SS needle complex. We then screened a collection of 146 Traditional Chinese Medicine extracts and identified potent inhibition of T3SS-dependent secretions of protein effectors. Based on the candidate search from the extracts, we found a novel class of T3SS inhibitors. This class of compounds has the capacity to modulate the secretion of many other Salmonella effectors. Moreover, it also inhibits the infection of salmonella in cells. This study provides a new platform for studying inhibitors of T3SS in Gram-negative bacteria and facilitates the development of new class of antivirulence agents to combat new pathogens.

Exploiting redundancy in the genetic code: control of pathogen virulence via computationally designed genetic material

J. Robert Coleman, PhD, Albert Einstein College of Medicine

The rational design and large-scale de novo synthesis of genetic material makes it possible to customize genes. Given that the genetic code is redundant there is great flexibility in how one can encode a gene at its nucleic acid level without changing the protein it produces. For example, a typical 300 amino acid protein can have up to 10¹⁵¹ encodings, yet the primary amino acid sequence is left unchanged. This flexibility allows for the rational design of the genome of a pathogen to achieve attenuated vaccine strains or antigens that will aid in the development of novel vaccines. Recently, a newly described method of gene manipulation that uses synthetic biology, computer-based gene design and de novo DNA synthesis was used to attenuate viral virulence. The work described herein describes the successful application of genetic manipulation to the problem of bacterial virulence by altering the amount of protein expression, thereby reducing pathogenicity.

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