Biocatalytic synthesis of non-vicinal aliphatic diols.

Biocatalysts are receiving increased attention in the field of selective oxyfunctionalization of C-H bonds, with cytochrome P450 monooxygenases (CYP450s), and the related peroxygenases, leading the field. Here we report on the substrate promiscuity of CYP505A30, previously characterized as a fatty acid hydroxylase. In addition to its regioselective oxyfunctionalization of saturated fatty acids (ω-1 - ω-3 hydroxylation), primary fatty alcohols are also accepted with similar regioselectivities. Moreover, alkanes such as n-octane and n-decane are also readily accepted, allowing for the production of non-vicinal diols through sequential oxygenation.

X-ray crystal structure and specificity of the Toxoplasma gondii ME49 TgAPN2.

Toxoplasmosis is a parasitic disease caused by infection with Toxoplasma gondii that currently has few therapeutic options. The M1 aminopeptidase enzymes have been shown to be attractive targets for anti-parasitic agents and/or vaccine candidates, suggesting potential to re-purpose inhibitors between parasite M1 aminopeptidase targets. The M1 aminopeptidase TgAPN2 has been suggested to be a potential new drug target for toxoplasmosis. Here we investigate the structure and function of TgAPN2, a homologue of the antimalarial drug target PfA-M1, and evaluate the capacity to use inhibitors that target PfA-M1 against TgAPN2. The results show that despite a similar overall fold, the TgAPN2 has a unique substrate specificity and inhibition profile. Sequence and structure differences are investigated and show how comparative structure-activity relationships may provide a route to obtaining potent inhibitors of TgAPN2.

Identifying novel chemical matter against the Chikungunya virus nsP3 macrodomain through crystallographic fragment screening.

Chikungunya virus (CHIKV) causes severe fever, rash and debilitating joint pain that can last for months1,2or even years. Millions of people have been infected with CHIKV, mostly in low and middle-income countries, and the virus continues to spread into new areas due to the geographical expansion of its mosquito hosts. Its genome encodes a macrodomain, which functions as an ADP-ribosyl hydrolase, removing ADPr from viral and host-cell proteins interfering with the innate immune response. Mutational studies have shown that the CHIKV nsP3 macrodomain is necessary for viral replication, making it a potential target for the development of antiviral therapeutics. We, therefore, performed a high-throughput crystallographic fragment screen against the CHIKV nsP3 macrodomain, yielding 109 fragment hits covering the ADPr-binding site and two adjacent subsites that are absent in the homologous macrodomain of SARS-CoV-2 but may be present in other alphaviruses, such as Venezuelan equine encephalitis virus (VEEV) and eastern equine encephalitis virus (EEEV). Finally, a subset of overlapping fragments was used to manually design three fragment merges covering the adenine and oxyanion subsites. The rich dataset of chemical matter and structural information discovered from this fragment screen is publicly available and can be used as a starting point for developing a CHIKV nsP3 macrodomain inhibitor.

Discovery of potent SARS-CoV-2 nsp3 macrodomain inhibitors uncovers lack of translation to cellular antiviral response.

A strategy for pandemic preparedness is the development of antivirals against a wide set of viral targets with complementary mechanisms of action. SARS-CoV-2 nsp3-mac1 is a viral macrodomain with ADP-ribosylhydrolase activity, which counteracts host immune response. Targeting the virus' immunomodulatory functionality offers a differentiated strategy to inhibit SARS-CoV-2 compared to approved therapeutics, which target viral replication directly. Here we report a fragment-based lead generation campaign guided by computational approaches. We discover tool compounds which inhibit nsp3-mac1 activity at low nanomolar concentrations, and with responsive structure-activity relationships, high selectivity, and drug-like properties. Using our inhibitors, we show that inhibition of nsp3-mac1 increases ADP-ribosylation, but surprisingly does not translate to demonstrable antiviral activity in cell culture and iPSC-derived pneumocyte models. Further, no synergistic activity is observed in combination with interferon gamma, a main protease inhibitor, nor a papain-like protease inhibitor. Our results question the extent to which targeting modulation of innate immunity-driven ADP-ribosylation can influence SARS-CoV-2 replication. Moreover, these findings suggest that nsp3-mac1 might not be a suitable target for antiviral therapeutics development.

Open science discovery of potent noncovalent SARS-CoV-2 main protease inhibitors.

We report the results of the COVID Moonshot, a fully open-science, crowdsourced, and structure-enabled drug discovery campaign targeting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease. We discovered a noncovalent, nonpeptidic inhibitor scaffold with lead-like properties that is differentiated from current main protease inhibitors. Our approach leveraged crowdsourcing, machine learning, exascale molecular simulations, and high-throughput structural biology and chemistry. We generated a detailed map of the structural plasticity of the SARS-CoV-2 main protease, extensive structure-activity relationships for multiple chemotypes, and a wealth of biochemical activity data. All compound designs (>18,000 designs), crystallographic data (>490 ligand-bound x-ray structures), assay data (>10,000 measurements), and synthesized molecules (>2400 compounds) for this campaign were shared rapidly and openly, creating a rich, open, and intellectual property-free knowledge base for future anticoronavirus drug discovery.