Understanding mechanisms of terpene synthases using substrate analogs.

Terpene synthases (TS) transform achiral prenyl substrates into elaborate hydrocarbon scaffolds with multiple stereocenters through a series of cyclization reactions and carbon skeleton rearrangements. The reactions involve high-energy carbocation intermediates that must be stabilized by the enzyme along the pathway to the desired products. A variety of substrate analogs have been used to investigate TS mechanism. This article will focus on a class of analogs which strategically replace hydrogen atoms with fluorine to inhibit the generation of specific carbocation intermediates. We will explore the synthesis and use of the analogs to study TS mechanism.

Crystal Structure of Caryolan-1-ol Synthase, a Sesquiterpene Synthase Catalyzing an Initial Anti-Markovnikov Cyclization Reaction.

In a continuing effort to understand reaction mechanisms of terpene synthases catalyzing initial anti-Markovnikov cyclization reactions, we solved the X-ray crystal structure of (+)-caryolan-1-ol synthase (CS) from Streptomyces griseus , with and without an inactive analog of the FPP substrate, 2-fluorofarnesyl diphosphate (2FFPP), bound in the active site of the enzyme. The CS-2FFPP complex was solved to 2.65 Å resolution and showed the ligand in a linear, elongated orientation, incapable of undergoing the initial cyclization event to form a bond between carbons C1 and C11. Intriguingly, the apo CS structure (2.2 Å) also had electron density in the active site, in this case density that was well fit with a curled-up tetraethylene glycol molecule presumably recruited from the crystallization medium. The density was also well fit by a molecule of farnesene suggesting that the structure may mimic an intermediate along the reaction coordinate. The curled-up conformation of tetraethylene glycol was accompanied by dramatic rotamer shifts among active-site residues. Most notably, W56 was observed to undergo a 90° rotation between the 2FFPP complex and apo-enzyme structures, suggesting that it contributes to steric interactions that help curl the tetraethylene glycol molecule in the active site, and by extension perhaps also a derivative of the FPP substrate in the normal course of the cyclization reaction. In support of this proposal, the CS W56L variant lost the ability to cyclize the FPP substrate and produced only the linear terpene products farnesol and α- and ß-farnesene.

Evaluation of SARS-CoV-2 Main Protease Inhibitors Using a Novel Cell-Based Assay.

As an essential enzyme of SARS-CoV-2, main protease (MPro) triggers acute toxicity to its human cell host, an effect that can be alleviated by an MPro inhibitor. Using this toxicity alleviation, we developed an effective method that allows a bulk analysis of the cellular potency of MPro inhibitors. This novel assay is advantageous over an antiviral assay in providing precise cellular MPro inhibition information to assess an MPro inhibitor. We used this assay to analyze 30 known MPro inhibitors. Contrary to their strong antiviral effects and up to 10 µM, 11a, calpain inhibitor II, calpain XII, ebselen, bepridil, chloroquine, and hydroxychloroquine showed relatively weak to undetectable cellular MPro inhibition potency implicating their roles in interfering with key steps other than just the MPro catalysis in the SARS-CoV-2 life cycle. Our results also revealed that MPI5, MPI6, MPI7, and MPI8 have high cellular and antiviral potency. As the one with the highest cellular and antiviral potency among all tested compounds, MPI8 has a remarkable cellular MPro inhibition IC50 value of 31 nM that matches closely to its strong antiviral effect with an EC50 value of 30 nM. Therefore, we cautiously suggest exploring MPI8 further for COVID-19 preclinical tests.

QUANTIFYING BILATERAL INFECTION PATTERNS IN THE TREMATODE ALLOGLOSSIDIUM RENALE.

Within-host distributions of parasites can have relevance to parasite competition, parasite mating, transmission, and host health. We examined the within-host distribution of the adult trematode Alloglossidium renale infecting the paired antennal glands of grass shrimp. There are 4 possible parasite distributions for infections of paired organs: random, uniform, biased aggregation to 1 particular organ (e.g., left vs. right), or inconsistently biased (aggregated, but does not favor 1 side). Previous work has shown that morphological asymmetries in hosts can lead to biased infections of paired organs. Apparent symmetry between the antennal glands of grass shrimp leads to the prediction that there would be no bias for 1 particular organ. However, an alternative prediction stems from the fact that A. renale is hermaphroditic: aggregation between glands would increase outcrossing opportunities and thus, avoid inbreeding via self-mating. Existing methods to test for an overall pattern did not apply to the A. renale system because of low-intensity infections as well as many 0 values for abundance per unit of the antennal gland. Hence, we used Monte Carlo simulations to determine if the observed overall patterns differed from those expected by randomly allocating parasites into groups of 2. We found that in 3 of 4 data sets, A. renale infections did not deviate from random distributions. The fourth data set had a more uniform pattern than expected by chance. As there was no aggregation between glands and the proportion of worms in single gland infections did not differ from that expected by chance alone, we found no evidence of inbreeding avoidance as might be manifested via a within-host distribution. Given the large proportion of worms in single infections, we predict as a major evolutionary outcome that populations of A. renale will be largely inbred.