Structure-activity relationships of a lead hydroxamic acid inhibitor of recombinant human stromelysin were systematically defined by taking advantage of a concise synthesis that allowed diverse functionality to be explored at each position in a template. An ex vivo rat model and an in vivo rabbit model of stromelysin-induced cartilage degradation were used to further optimize these analogs for oral activity and duration of action. The culmination of these modifications resulted in CGS 27023A, a potent, orally active stromelysin inhibitor that blocks the erosion of cartilage matrix.

Synthetic lethal screening is a chemical biology approach to identify small molecules that selectively kill oncogene-expressing cell lines with the goal of identifying pathways that provide specific targets against cancer cells. We performed a high-throughput screen of 303,282 compounds from the National Institutes of Health-Molecular Libraries Small Molecule Repository (NIH-MLSMR) against immortalized BJ fibroblasts expressing HRASG12V followed by a counterscreen of lethal compounds in a series of isogenic cells lacking the HRASG12V oncogene. This effort led to the identification of two novel molecular probes (PubChem CID 3689413, ML162 and CID 49766530, ML210) with nanomolar potencies and 4–23 fold selectivities, which can potentially be used for identifying oncogene-specific pathways and targets in cancer cells.

DNA gyrase is an essential enzyme in bacteria, and its inhibition results in the disruption of DNA synthesis and, subsequently, cell death. The pyrrolamides are a novel class of antibacterial agents targeting DNA gyrase. These compounds were identified by a fragment-based lead generation (FBLG) approach using nuclear magnetic resonance (NMR) screening to identify low-molecular-weight compounds that bind to the ATP pocket of DNA gyrase. A pyrrole hit with a binding constant of 1 mM formed the basis of the design and synthesis of a focused library of compounds that resulted in the rapid identification of a lead compound that inhibited DNA gyrase with a 50% inhibitory concentration (IC(50)) of 3 μM. The potency of the lead compound was further optimized by utilizing iterative X-ray crystallography to yield DNA gyrase inhibitors that also displayed antibacterial activity. Spontaneous mutants were isolated in Staphylococcus aureus by plating on agar plates containing pyrrolamide 4 at the MIC. The resistant variants displayed 4- to 8-fold-increased MIC values relative to the parent strain. DNA sequencing revealed two independent point mutations in the pyrrolamide binding region of the gyrB genes from these variants, supporting the hypothesis that the mode of action of these compounds was inhibition of DNA gyrase. Efficacy of a representative pyrrolamide was demonstrated against Streptococcus pneumoniae in a mouse lung infection model. These data demonstrate that the pyrrolamides are a novel class of DNA gyrase inhibitors with the potential to deliver future antibacterial agents targeting multiple clinical indications.

A high-throughput screen of the NIH-MLSMR compound collection, along with a series of secondary assays to identify potential targets of hit compounds, previously identified a 1,3-diaminobenzene scaffold that targets protease-activated receptor 1 (PAR1). We now report additional structure–activity relationship (SAR) studies that delineate the requirements for activity at PAR1 and identify plasma-stable analogues with nanomolar inhibition of PAR1-mediated platelet activation. Compound 4 was declared as a probe (ML161) with the NIH Molecular Libraries Program. This compound inhibited platelet aggregation induced by a PAR1 peptide agonist or by thrombin but not by several other platelet agonists. Initial studies suggest that ML161 is an allosteric inhibitor of PAR1. These findings may be important for the discovery of antithrombotics with an improved safety profile.

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A detailed study of the chemical behavior of modern catalysts (here, exemplified by dehydrogenases dependent on NAD+) allows us to construct models that distinguish between selected and drifting behaviors in biological macromolecules. These models enable us to manipulate rationally the properties of enzymes, here to design an "acetaldehyde reductase" dependent on NAD+ that is faster than any given us by nature. When applied to the origin of protein catalysis, models that explain the structures of ribo-cofactors (e.g., NAD+) must postulate a metabolically complex breakthrough organism. This means that: (1) The view from the present day back to the truly primeval organism is obscured; it is futile to try to deduce the detailed structure of the first life by examining the behaviors of modern organisms. (2) Riboorganisms dominated life on earth for a long time before translation evolved; indeed, fossils of riboorganisms might already be known. (3) Using organic synthesis, we have expanded the number of bases available for making RNA and making accessible RNA molecules that are likely to be intrinsically better catalysts.

A research program has applied the tools of synthetic organic chemistry to systematically modify the structure of DNA and RNA oligonucleotides to learn more about the chemical principles underlying their ability to store and transmit genetic information. Oligonucleotides (as opposed to nucleosides) have long been overlooked by synthetic organic chemists as targets for structural modification. Synthetic chemistry has now yielded oligonucleotides with 12 replicatable letters, modified backbones, and new insight into why Nature chose the oligonucleotide structures that she did.The "standard model" of nucleic acid structure dates back to 1953 and two classic papers by Watson and Crick.132 It has been little altered since. The model holds that the energy of binding of two complementdry DNA or RNA (oligonucleotide) strands arises from the stacking of the hydrophobic nucleobases, while the specificity of the association arises from base pairing following two simple rules ("A pairs with T, G pairs with C"). No other class of natural products has reactivity that obeys such simple rules. Nor is it obvious how one designs a class of chemical substances that does so much so simply. Despite this chemical conundrum, and the position of nucleic acids at the center of natural product chemistry, few organic chemists have chosen to apply their synthetic skills to explore reactivity at the level of the oligonucleotide. Much work had been done, of course, in making structurally modified analogs of nucleosides, both in industry and academia.3 But most organic chemists, attracted by the structural intricacies of secondary metabolites, have neglected oligonucleotides as targets for structural modification.Some 15 years ago we began a program to fill this gap, developing synthetic organic chemistry and organic structural theory as it applies to nucleic acids in their oligomeric form. This began with one of the first two total syntheses of a gene encoding a p r~t e i n ,~ and has continued with the development of structurally altered oligonucleotides. As in all organic chemistry that alters the structure of natural products, our goal has been to learn more about how DNA and RNA work. We focus here on chemistry that has modified the bases, the sugars, and the backbones of oligonucleotides.

A potent macrocyclic inhibitor of neutral endopeptidase (NEP) 24.11 was designed using a computer model of the active site of thermolysin. This 10-membered ring lactam represents a general mimic for any hydrophobic dipeptide in which the two amino acid side chains bind to an enzyme in a contiguous orientation. The parent 10-membered ring lactam was synthesized and exhibited excellent potency as an NEP 24.11 inhibitor (IC50 = 3 nM). In order to improve oral bioavailability, various functionality was attached to the macrocycle. These modifications lead to CGS 25155, an orally active NEP 24.11 inhibitor that slows down the degradation of the cardiac hormone atrial natriuretic factor, producing a lowering of blood pressure in the DOCA-salt rat model of hypertension.