Human rhinoviruses, the most important etiologic agents of the common cold, are messenger-active single-stranded monocistronic RNA viruses that have evolved a highly complex cascade of proteolytic processing events to control viral gene expression and replication. Most maturation cleavages within the precursor polyprotein are mediated by rhinovirus 3C protease (or its immediate precursor, 3CD), a cysteine protease with a trypsin-like polypeptide fold. Highresolution crystal structures of the enzyme from three viral serotypes have been used for the design and elaboration of 3C protease inhibitors representing different structural and chemical classes. Inhibitors having ␣,␤-unsaturated carbonyl groups combined with peptidyl-binding elements specific for 3C protease undergo a Michael reaction mediated by nucleophilic addition of the enzyme's catalytic Cys-147, resulting in covalent-bond formation and irreversible inactivation of the viral protease. Direct inhibition of 3C proteolytic activity in virally infected cells treated with these compounds can be inferred from dose-dependent accumulations of viral precursor polyproteins as determined by SDS͞PAGE analysis of radiolabeled proteins. Cocrystal-structure-assisted optimization of 3C-protease-directed Michael acceptors has yielded molecules having extremely rapid in vitro inactivation of the viral protease, potent antiviral activity against multiple rhinovirus serotypes and low cellular toxicity. Recently, one compound in this series, AG7088, has entered clinical trials.

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The structure-based design, chemical synthesis, and biological evaluation of various human rhinovirus (HRV) 3C protease (3CP) inhibitors which incorporate P1 lactam moieties in lieu of an L-glutamine residue are described. These compounds are comprised of a tripeptidyl or peptidomimetic binding determinant and an ethyl propenoate Michael acceptor moiety which forms an irreversible covalent adduct with the active site cysteine residue of the 3C enzyme. The P1-lactam-containing inhibitors display significantly increased 3CP inhibition activity along with improved antirhinoviral properties relative to corresponding L-glutamine-derived molecules. In addition, several lactam-containing compounds exhibit excellent selectivity for HRV 3CP over several other serine and cysteine proteases and are not appreciably degraded by a variety of biological agents. One of the most potent inhibitors (AG7088, mean antirhinoviral EC90 approximately 0.10 microM, n = 46 serotypes) is shown to warrant additional preclinical development to explore its potential for use as an antirhinoviral agent.

The ability to selectively degrade proteins with bifunctional small molecules has the potential to fundamentally alter therapy in a variety of diseases. However, the relatively large size of these chimeric molecules often results in challenging physico‐chemical properties (e. g., low aqueous solubility) and poor pharmacokinetics which may complicate their in vivo applications. We recently discovered an exquisitely potent chimeric BET degrader (GNE‐987) which exhibited picomolar cell potencies but also demonstrated low in vivo exposures. In an effort to improve the pharmacokinetic properties of this molecule, we discovered the first degrader‐antibody conjugate by attaching GNE‐987 to an anti‐CLL1 antibody via a novel linker. A single IV dose of the conjugate afforded sustained in vivo exposures that resulted in antigen‐specific tumor regressions. Enhancement of a chimeric protein degrader with poor in vivo properties through antibody conjugation thereby expands the utility of directed protein degradation as both a biological tool and a therapeutic possibility.

170215-E130/91) and Gobierno Vasco (Project PGV: 9113.1). Supplementary Material Available: Listings of experimental details and spectral data for 4a-d, 7a,b, 8a-d, lla-d, 14a-d, 15a, 16a, 22, 23, 28, and 29, tables of X-ray data and structures for 6a, 38, and 39, and NMR spectra for 4c, 7a,b, and 19-21 (18 pages); table of observed and calculated structure factors (19 pages). Ordering information is given on any current masthead page.Studies on the Thermal Generation and Reactivity of a Class of ( , )-1,4-Biradicals