Matthew C. T. Hartman

In Vitro Selection of Highly Modified Cyclic Peptides That Act as Tight Binding Inhibitors

There is a great demand for the discovery of new therapeutic molecules that combine the high specificity and affinity of biologic drugs with the bioavailability and lower cost of small molecules. Small, natural-product-like peptides hold great promise in bridging this gap; however, access to libraries of these compounds has been a limitation. Since ribosomal peptides may be subjected to in vitro selection techniques, the generation of extremely large libraries (>1013) of highly modified macrocyclic peptides may provide a powerful alternative for the generation and selection of new useful bioactive molecules. Moreover, the incorporation of many non-proteinogenic amino acids into ribosomal peptides in conjunction with macrocyclization should enhance the drug-like features of these libraries. Here we show that mRNA-display, a technique that allows the in vitro selection of peptides, can be applied to the evolution of macrocyclic peptides that contain a majority of unnatural amino acids. We describe the isolation and characterization of two such unnatural cyclic peptides that bind the protease thrombin with low nanomolar affinity, and we show that the unnatural residues in these peptides are essential for the observed high-affinity binding. We demonstrate that the selected peptides are tight-binding inhibitors of thrombin, with Kiapp values in the low nanomolar range. The ability to evolve highly modified macrocyclic peptides in the laboratory is the first crucial step toward the facile generation of useful molecular reagents and therapeutic lead molecules that combine the advantageous features of biologics with those of small-molecule drugs.

Ribosomal Synthesis of N-Methyl Peptides

N-Methyl amino acids (N-Me AAs) are a common component of nonribosomal peptides (NRPs), a class of natural products from which many clinically important therapeutics are obtained. N-Me AAs confer peptides with increased conformational rigidity, membrane permeability, and protease resistance. Hence, these analogues are highly desirable building blocks in the ribosomal synthesis of unnatural peptide libraries, from which functional, NRP-like molecules may be identified. By supplementing a reconstituted Escherichia coli translation system with specifically aminoacylated total tRNA that has been chemically methylated, we have identified three N-Me AAs (N-Me Leu, N-Me Thr, and N-Me Val) that are efficiently incorporated into peptides by the ribosome. Moreover, we have demonstrated the synthesis of peptides containing up to three N-Me AAs, a number comparable to that found in many NRP drugs. With improved incorporation efficiency and translational fidelity, it may be possible to synthesize combinatorial libraries of peptides that contain multiple N-Me AAs. Such libraries could be subjected to in vitro selection methods to identify drug-like, high-affinity ligands for protein targets of interest.

A new, highly water-soluble, fluorescent turn-on chemodosimeter for direct measurement of hydrogen sulfide in biological fluids

A new reaction-based fluorescent reporter for H(2)S has been developed based on 8-aminopyrene-1,3,6-trisulfonate. This reporter shows high selectivity for H(2)S over other ions and thiols, and can detect H(2)S directly in serum without additives.

BRCA1-directed, enhanced and aberrant homologous recombination: mechanism and potential treatment strategies.

Despite intense studies, questions still remain regarding the molecular mechanisms leading to the development of hereditary breast and ovarian cancers. Research focused on elucidating the role of the breast cancer susceptibility gene 1 (BRCA1) in the DNA damage response may be of the most critical importance to understanding these processes. The BRCA1 protein has an N-terminal RING domain possessing E3 ubiquitinligase activity and a C-terminal BRCT domain involved in binding specific phosphoproteins. These domains are involved directly or indirectly in DNA double-strand break (DSB) repair. As the two terminal domains of BRCA1 represent two separate entities, understanding how these domains communicate and are functionally altered in regards to DSB repair is critical for understanding the development of BRCA1-related breast and ovarian cancers and for developing novel therapeutics. Herein, we review recent findings of how altered functions of these domains might lead to cancer through a mechanism of increased aberrant homologous recombination and possible implications for the development of BRCA1 inhibitors.

Photocaged permeability: a new strategy for controlled drug release

Light is used to release a drug from a cell impermeable small molecule, uncloaking its cytotoxic effect on cancer cells.

Optimal Codon Choice Can Improve the Efficiency and Fidelity of N- Methyl Amino Acid Incorporation into Peptides by In-Vitro Translation**

premature termination of translation can occur or incorrect amino acids can be misincorporated in place of the desired NMe AA, resulting in reduced incorporation efficiency and fidelity of the N-Me AA. We sought to determine the main cause of such misincorporation and truncation events, and then to optimize the incorporation of N-Me AAs. We examined the efficiency and fidelity with which three different N-Me AAs (N-Me Leu, N-Me Thr, and N-Me Val) are incorporated at a single position in a short peptide (MH6MXmEP, Xm = N-Me AA, M = Met, E = Glu, P = Pro, H = His) using each of the codons of the corresponding natural amino acid (Figure 1). We showed previously that these three N-Me AAs can be incorporated into peptides by in-vitro translation [11] using the fully reconstituted PURE system for translation (PURE = protein synthesis using recombinant elements, an in vitro system containing only purifies components). [13] We directed the incorporation of these N-Me AAs into peptides by supplying in-vitro translation reaction mixtures with total tRNA that had been enzymatically precharged with Leu, Thr, or Val and then chemically N-methylated as previously described (see Experimental Section). [7, 11] The presence of the other 19 amino acid/ aminoacyl-tRNA synthetase (AARS) pairs in the translation

Synthesis of Novel Peptide Linkers: Simultaneous Cyclization and Labeling

Synthesis of novel peptide linkers was accomplished by monocarboxylation of 1,3,5-tris(bomomethyl)benzene with a wide variety of carboxylic acids in the presence of diisopropylethylamine. These reagents can be used to simultaneously cyclize and label peptides containing two cysteines. Many labels are compatible with this method including lipids, fluorescent groups, and biotin.

Highly Constrained Bicyclic Scaffolds for the Discovery of Protease-Stable Peptides via mRNA Display

Highly constrained peptides such as the knotted peptide natural products are promising medicinal agents because of their impressive biostability and potent activity. Yet, libraries of highly constrained peptides are challenging to prepare. Here, we present a method which utilizes two robust, orthogonal chemical steps to create highly constrained bicyclic peptide libraries. This technology was optimized to be compatible with in vitro selections by mRNA display. We performed side-by-side monocyclic and bicyclic selections against a model protein (streptavidin). Both selections resulted in peptides with mid-nanomolar affinity, and the bicyclic selection yielded a peptide with remarkable protease resistance.

In Vitro Selection of Unnatural Cyclic Peptide Libraries via mRNA Display

The ribosomal synthesis of drug-like peptides containing unnatural amino acids is possible due to the broad substrate specificity of the ribosome. In this protocol, a reconstituted Escherichia coli ribosomal translation system (PURE) is adapted to incorporate unnatural amino acids into mRNA-displayed peptide libraries, which are used in in vitro selection.

Peptide library approach to uncover phosphomimetic inhibitors of the BRCA1 C-terminal domain.

Many intracellular protein-protein interactions are mediated by the phosphorylation of serine, and phosphoserine-containing peptides can inhibit these interactions. However, hydrolysis of the phosphate by phosphatases, and the poor cell permeability associated with phosphorylated peptides has limited their utility in cellular and in vivo contexts. Compounding the problem, strategies to replace phosphoserine in peptide inhibitors with easily accessible mimetics (such as Glu or Asp) routinely fail. Here, we present an in vitro selection strategy for replacement of phosphoserine. Using mRNA display, we created a 10 trillion member structurally diverse unnatural peptide library. From this library, we found a peptide that specifically binds to the C-terminal domain (BRCT)2 of breast cancer associated protein 1 (BRCA1) with an affinity comparable to phosphorylated peptides. A crystal structure of the peptide bound reveals that the pSer-x-x-Phe motif normally found in BRCA1 (BRCT)2 binding partners is replaced by a Glu-x-x-4-fluoroPhe and that the peptide picks up additional contacts on the protein surface not observed in cognate phosphopeptide binding. Expression of the peptide in human cells led to defects in DNA repair by homologous recombination, a process BRCA1 is known to coordinate. Overall, this work validates a new in vitro selection approach for the development of inhibitors of protein-protein interactions mediated by serine phosphorylation.