Paolo A. Centrella

Design, synthesis and selection of DNA-encoded small-molecule libraries.

Biochemical combinatorial techniques such as phage display, RNA display and oligonucleotide aptamers have proven to be reliable methods for generation of ligands to protein targets. Adapting these techniques to small synthetic molecules has been a long-sought goal. We report the synthesis and interrogation of an 800-million-member DNA-encoded library in which small molecules are covalently attached to an encoding oligonucleotide. The library was assembled by a combination of chemical and enzymatic synthesis, and interrogated by affinity selection. We describe methods for the selection and deconvolution of the chemical display library, and the discovery of inhibitors for two enzymes: Aurora A kinase and p38 MAP kinase.

Discovery of Highly Potent and Selective Small Molecule ADAMTS-5 Inhibitors That Inhibit Human Cartilage Degradation via Encoded Library Technology (ELT)

The metalloprotease ADAMTS-5 is considered a potential target for the treatment of osteoarthritis. To identify selective inhibitors of ADAMTS-5, we employed encoded library technology (ELT), which enables affinity selection of small molecule binders from complex mixtures by DNA tagging. Selection of ADAMTS-5 against a four-billion member ELT library led to a novel inhibitor scaffold not containing a classical zinc-binding functionality. One exemplar, (R)-N-((1-(4-(but-3-en-1-ylamino)-6-(((2-(thiophen-2-yl)thiazol-4-yl)methyl)amino)-1,3,5-triazin-2-yl)pyrrolidin-2-yl)methyl)-4-propylbenzenesulfonamide (8), inhibited ADAMTS-5 with IC(50) = 30 nM, showing >50-fold selectivity against ADAMTS-4 and >1000-fold selectivity against ADAMTS-1, ADAMTS-13, MMP-13, and TACE. Extensive SAR studies showed that potency and physicochemical properties of the scaffold could be further improved. Furthermore, in a human osteoarthritis cartilage explant study, compounds 8 and 15f inhibited aggrecanase-mediated (374)ARGS neoepitope release from aggrecan and glycosaminoglycan in response to IL-1β/OSM stimulation. This study provides the first small molecule evidence for the critical role of ADAMTS-5 in human cartilage degradation.

Encoded Library Technology as a Source of Hits for the Discovery and Lead Optimization of a Potent and Selective Class of Bactericidal Direct Inhibitors of Mycobacterium tuberculosis InhA

Tuberculosis (TB) is one of the worlds oldest and deadliest diseases, killing a person every 20 s. InhA, the enoyl-ACP reductase from Mycobacterium tuberculosis, is the target of the frontline antitubercular drug isoniazid (INH). Compounds that directly target InhA and do not require activation by mycobacterial catalase peroxidase KatG are promising candidates for treating infections caused by INH resistant strains. The application of the encoded library technology (ELT) to the discovery of direct InhA inhibitors yielded compound 7 endowed with good enzymatic potency but with low antitubercular potency. This work reports the hit identification, the selected strategy for potency optimization, the structure-activity relationships of a hundred analogues synthesized, and the results of the in vivo efficacy studies performed with the lead compound 65.

Encoded Library Synthesis Using Chemical Ligation and the Discovery of sEH Inhibitors from a 334-Million Member Library

A chemical ligation method for construction of DNA-encoded small-molecule libraries has been developed. Taking advantage of the ability of the Klenow fragment of DNA polymerase to accept templates with triazole linkages in place of phosphodiesters, we have designed a strategy for chemically ligating oligonucleotide tags using cycloaddition chemistry. We have utilized this strategy in the construction and selection of a small molecule library, and successfully identified inhibitors of the enzyme soluble epoxide hydrolase.

Carbamate Analogues of Fumagillin as Potent, Targeted Inhibitors of Methionine Aminopeptidase-2

Inhibition of methionine aminopeptidase-2 (MetAP2) represents a novel approach to antiangiogenic therapy. We describe the synthesis and activity of fumagillin analogues that address the pharmacokinetic and safety liabilities of earlier candidates in this compound class. Two-step elaboration of fumagillol with amines yielded a diverse series of carbamates at C6 of the cyclohexane spiroepoxide. The most potent of these compounds exhibited subnanomolar inhibition of cell proliferation in HUVEC and BAEC assays. Although a range of functionalities were tolerated at this position, alpha-trisubstituted amines possessed markedly decreased inhibitory activity, and this could be rationalized by modeling based on the known fumagillin-MetAP2 crystal structure. The lead compound resulting from these studies, (3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-enyl)oxiran-2-yl)-1-oxaspiro[2.5]octan-6-yl (R)-1-amino-3-methyl-1-oxobutan-2-ylcarbamate, (PPI-2458), demonstrated an improved pharmacokinetic profile relative to the earlier clinical candidate TNP-470, and has advanced into phase I clinical studies in non-Hodgkins lymphoma and solid cancers.

Cell-Based Selection Expands the Utility of DNA-Encoded Small-Molecule Library Technology to Cell Surface Drug Targets: Identification of Novel Antagonists of the NK3 Tachykinin Receptor

DNA-encoded small-molecule library technology has recently emerged as a new paradigm for identifying ligands against drug targets. To date, this technology has been used with soluble protein targets that are produced and used in a purified state. Here, we describe a cell-based method for identifying small-molecule ligands from DNA-encoded libraries against integral membrane protein targets. We use this method to identify novel, potent, and specific inhibitors of NK3, a member of the tachykinin family of G-protein coupled receptors (GPCRs). The method is simple and broadly applicable to other GPCRs and integral membrane proteins. We have extended the application of DNA-encoded library technology to membrane-associated targets and demonstrate the feasibility of selecting DNA-tagged, small-molecule ligands from complex combinatorial libraries against targets in a heterogeneous milieu, such as the surface of a cell.

Discovery of a Potent Class of PI3Kα Inhibitors with Unique Binding Mode via Encoded Library Technology (ELT)

In the search of PI3K p110α wild type and H1047R mutant selective small molecule leads, an encoded library technology (ELT) campaign against the desired target proteins was performed which led to the discovery of a selective chemotype for PI3K isoforms from a three-cycle DNA encoded library. An X-ray crystal structure of a representative inhibitor from this chemotype demonstrated a unique binding mode in the p110α protein.

Discovery of a Potent BTK Inhibitor with a Novel Binding Mode by Using Parallel Selections with a DNA‐Encoded Chemical Library

We have identified and characterized novel potent inhibitors of Brutons tyrosine kinase (BTK) from a single DNA‐encoded library of over 110 million compounds by using multiple parallel selection conditions, including variation in target concentration and addition of known binders to provide competition information. Distinct binding profiles were observed by comparing enrichments of library building block combinations under these conditions; one enriched only at high concentrations of BTK and was competitive with ATP, and another enriched at both high and low concentrations of BTK and was not competitive with ATP. A compound representing the latter profile showed low nanomolar potency in biochemical and cellular BTK assays. Results from kinetic mechanism of action studies were consistent with the selection profiles. Analysis of the co‐crystal structure of the most potent compound demonstrated a novel binding mode that revealed a new pocket in BTK. Our results demonstrate that profile‐based selection strategies using DNA‐encoded libraries form the basis of a new methodology to rapidly identify small molecule inhibitors with novel binding modes to clinically relevant targets.

Discovery of cofactor-specific, bactericidal Mycobacterium tuberculosis InhA inhibitors using DNA-encoded library technology

Significance The increasing prevalence of multidrug-resistant strains of tuberculosis has created an urgent need for novel therapies to treat tuberculosis infections. Here we have demonstrated the successful utilization of the DNA-encoded X-Chem technology for the discovery inhibitors of Mycobacterium tuberculosis enoyl–acyl-carrier protein (ACP) reductase, InhA, a validated target for the treatment of tuberculosis. The identified inhibitors are cofactor specific and have activity in multiple cellular assays. Crystal structures of representative compounds from five chemical series revealed that the compounds bind adjacent to the NADH cofactor and adopt a variety of conformations, including two previously unreported binding modes. The compounds identified may serve as useful leads in the development of new antibacterial drugs with efficacy against multidrug-resistant tuberculosis. Millions of individuals are infected with and die from tuberculosis (TB) each year, and multidrug-resistant (MDR) strains of TB are increasingly prevalent. As such, there is an urgent need to identify novel drugs to treat TB infections. Current frontline therapies include the drug isoniazid, which inhibits the essential NADH-dependent enoyl–acyl-carrier protein (ACP) reductase, InhA. To inhibit InhA, isoniazid must be activated by the catalase-peroxidase KatG. Isoniazid resistance is linked primarily to mutations in the katG gene. Discovery of InhA inhibitors that do not require KatG activation is crucial to combat MDR TB. Multiple discovery efforts have been made against InhA in recent years. Until recently, despite achieving high potency against the enzyme, these efforts have been thwarted by lack of cellular activity. We describe here the use of DNA-encoded X-Chem (DEX) screening, combined with selection of appropriate physical properties, to identify multiple classes of InhA inhibitors with cell-based activity. The utilization of DEX screening allowed the interrogation of very large compound libraries (1011 unique small molecules) against multiple forms of the InhA enzyme in a multiplexed format. Comparison of the enriched library members across various screening conditions allowed the identification of cofactor-specific inhibitors of InhA that do not require activation by KatG, many of which had bactericidal activity in cell-based assays.

Antiparasitic Activities of Novel, Orally Available Fumagillin Analogs

Fumagillin, an irreversible inhibitor of MetAP2, has been shown to potently inhibit growth of malaria parasites in vitro. Here, we demonstrate activity of fumagillin analogs with an improved pharmacokinetic profile against malaria parasites, trypanosomes, and amoebas. A subset of the compounds showed efficacy in a murine malaria model. The observed SAR forms a basis for further optimization of fumagillin based inhibitors against parasitic targets by inhibition of MetAP2.