The Rosetta software suite for macromolecular modeling, docking, and design is widely used in pharmaceutical, industrial, academic, non-profit, and government laboratories. Despite its broad modeling capabilities, Rosetta remains consistently among leading software suites when compared to other methods created for highly specialized protein modeling and design tasks. Developed for over two decades by a global community of over 60 laboratories, Rosetta has undergone multiple refactorings, and now comprises over three million lines of code. Here we discuss methods developed in the last five years in Rosetta, involving the latest protocols for structure prediction; protein-protein and protein-small molecule docking; protein structure and interface design; loop modeling; the incorporation of various types of experimental data; modeling of peptides, antibodies and proteins in the immune system, nucleic acids, non-standard chemistries, carbohydrates, and membrane proteins. We briefly discuss improvements to the energy function, user interfaces, and usability of the software. Rosetta is available at www.rosettacommons.org.

Summary Naturally occurring, pharmacologically active peptides constrained with covalent crosslinks generally have shapes evolved to fit precisely into binding pockets on their targets. Such peptides can have excellent pharmaceutical properties, combining the stability and tissue penetration of small molecule drugs with the specificity of much larger protein therapeutics. The ability to design constrained peptides with precisely specified tertiary structures would enable the design of shape-complementary inhibitors of arbitrary targets. Here we describe the development of computational methods for de novo design of conformationally-restricted peptides, and the use of these methods to design 15–50 residue disulfide-crosslinked and heterochiral N-C backbone-cyclized peptides. These peptides are exceptionally stable to thermal and chemical denaturation, and twelve experimentally-determined X-ray and NMR structures are nearly identical to the computational models. The computational design methods and stable scaffolds presented here provide the basis for development of a new generation of peptide-based drugs.

We developed a de novo protein design strategy to swiftly engineer decoys for neutralizing pathogens that exploit extracellular host proteins to infect the cell. Our pipeline allowed the design, validation, and optimization of de novo hACE2 decoys to neutralize SARS-CoV-2. The best decoy, CTC-445.2, binds with low nanomolar affinity and high specificity to the RBD of the spike protein. Cryo-EM shows that the design is accurate and can simultaneously bind to all three RBDs of a single spike protein. Because the decoy replicates the spike protein target interface in hACE2, it is intrinsically resilient to viral mutational escape. A bivalent decoy, CTC-445.2d, shows ~10-fold improvement in binding. CTC-445.2d potently neutralizes SARS-CoV-2 infection of cells in vitro and a single intranasal prophylactic dose of decoy protected Syrian hamsters from a subsequent lethal SARS-CoV-2 challenge.

The pentein superfamily is a mechanistically diverse superfamily encompassing both noncatalytic proteins and enzymes that catalyze hydrolase, dihydrolase and amidinotransfer reactions on guanidine substrates. Despite generally low sequence identity, they possess a conserved structural fold and display common mechanistic themes in catalysis. The structurally characterized catalytic penteins possess a conserved core of residues that include a Cys, His and two polar, guanidine-binding residues. All known catalytic penteins use the core Cys to attack the substrate’s guanidine moiety to form a covalent thiouronium adduct and all cleave one or more of the guanidine C–N bonds. The mechanistic information compiled to date supports the hypothesis that this superfamily may have evolved divergently from a catalytically promiscuous ancestor.

In an effort to develop novel covalent modifiers of dimethylarginine dimethylaminohydrolase (DDAH) that are useful for biological applications, a set of "fragment"-sized inhibitors that were identified using a high-throughput screen are tested for time-dependent inhibition. One structural class of inactivators, 4-halopyridines, show time-and concentration-dependent inactivation of DDAH and the inactivation mechanism of one example, 4-bromo-2-methylpyridine (1), is characterized in detail. The neutral form of halopyridines is not very reactive with excess glutathione. However, 1 readily reacts, with loss of its halide, in a selective, covalent and irreversible manner with the active-site Cys249 of DDAH. This active-site Cys is not particularly reactive (pK a ca. 8.8) and 1 does not inactivate papain (Cys pK a ca. ≤ 4), suggesting that, unlike many reagents, Cys nucleophilicity is not a predominating factor in selectivity. Rather, binding and stabilization of the more reactive pyridinium form of the inactivator by a second moiety, Asp66, is required for facile reaction. This constraint imparts a unique selectivity profile to these inactivators. To our knowledge, halopyridines have not previously been reported as protein modifiers, and therefore represent a first-in-class example of a novel type of quiescent affinity label.

Small molecules capable of selective covalent protein modification are of significant interest for the development of biological probes and therapeutics. We recently reported that 2-methyl-4-bromopyridine is a quiescent affinity label for the nitric oxide controlling enzyme dimethylarginine dimethylaminohydrolase (DDAH) Am. Chem. Soc. 133, 1553-1562. Discovery of this novel protein modifier raised the possibility that the 4-halopyridine motif may be suitable for wider application. Therefore, the inactivation mechanism of the related compound 2-hydroxymethyl-4-chloropyridine is probed here in more detail. Solution studies support an inactivation mechanism in which the active-site Asp66 residue stabilizes the pyridinium form of the inactivator, which has enhanced reactivity toward the active site Cys, resulting in covalent bond formation, loss of the halide, and irreversible inactivation. A 2.18 Å resolution X-ray crystal structure of the inactivated complex elucidates the orientation of the inactivator and its covalent attachment to the active-site Cys, but the structural model does not show an interaction between the inactivator and Asp66. Molecular modeling is used to investigate inactivator binding, reaction, and also a final pyridinium deprotonation step that accounts for the apparent differences between the solution-based and structural studies with respect to the role of Asp66. This work integrates multiple approaches to elucidate the inactivation mechanism of a novel 4-halopyridine "warhead," emphasizing the strategy of using pyridinium formation as a "switch" to enhance reactivity when bound to the target protein.[Small molecules that are capable of covalently modifying proteins are currently undergoing a type of renaissance as they are newly applied to solve problems in chemical biology and proteomics, as well as in drug design, discovery and development. [1][2][3] There are a number of strategies that incorporate moderately electrophilic groups into the design of affinity-based probes, activity-based probes and activity-based protein profiling reagents. A few noted examples include the use of electrophilic phosphonates to target serine hydrolases, 4 * To whom correspondence should be addressed. W.F.: College of Pharmacy, PHAR-MED CHEM, 1 University Station; C0850, Austin, Texas 78712; Phone: (512) Fax: (512) During an effort to find novel inhibitors of the nitric oxide-controlling enzyme dimethylarginine dimethylaminohydrolase (DDAH), 11 2-methyl-4-bromopyridine was discovered to be a time-dependent inhibitor. 12 Mechanistic analysis determined that this 4-halopyridine is a covalent inactivator that selectively modifies the active-site Cys residue. The inactivation mechanism is most consistent with that described for quiescent affinity labels 13,14 in that the compound is relatively unreactive (quiescent) to most biological nucleophiles, but demonstrates an enhanced reactivity when bound to the active site of the target enzyme. In the specific case of 2-methyl-4-bromopyridine, DDAH was pro...

Inhibitors of human dimethylarginine dimethylaminohydrolase-1 (DDAH-1) are of therapeutic interest for controlling pathological nitric oxide production. Only a limited number of biologically useful inhibitors have been identified, so structurally diverse lead compounds are desired. In contrast with previous assays that do not possesses adequate sensitivity for optimal screening, herein is reported a high-throughput assay that uses an alternative thiol-releasing substrate, S-methyl-L-thiocitrulline, and a thiol-reactive fluorophore, 7-diethylamino-3-(4′-maleimidylphenyl)-4-methylcoumarin, to enable continuous detection of product formation by DDAH-1. The assay is applied to query two commercial libraries totaling 4,446 compounds and two representative hits are described, including a known DDAH-1 inhibitor. This is the most sensitive DDAH-1 assay reported to date, and enables screening of compound libraries using [S] =KM conditions, while displaying Z′ factors from 0.6 – 0.8. Therefore, this strategy now makes possible high-throughput screening for human DDAH-1 inhibitors in pursuit of molecular probes and drugs to control excessive nitric oxide production.

Dimethylarginine dimethylaminohydrolase (DDAH) is an endogenous regulator of nitric oxide production and represents a potential therapeutic target. However, only a small number of biologically useful inhibitors have been reported, and many of these are substrate analogs. To seek more diverse scaffolds, we developed a high-throughput screening (HTS) assay and queried two small libraries totaling 2446 compounds. The HTS assay proved to be robust, reproducible and scalable, with Z' factors ≥ 0.78. One inhibitor, ebselen, is structurally divergent from substrate and was characterized in detail. This selenazole covalently inactivates DDAH in vitro and in cultured cells. The rate constant for inactivation of DDAH (44,000 ± 2,400 M−1s−1) is greater than those reported for any other target, suggesting this pathway is an important aspect of ebselen's total pharmacological effects.