The growing defenses of the tuberculosis-causing bacteria against the body's immune system is the reason why they are so good at colonizing the human body .
‘The engineered compound that rendered the bacterium susceptible to nitric oxide, could be part of the new drug strategy for treating tuberculosis.’
"Given the increasing resistance ofáMycobacterium tuberculosisáto drugs, we contemplated the treatment of tuberculosis in a fundamentally different way," said Jason Sello, associate professor of chemistry at Brown who directed the research.
"Instead of seeking conventional drug leads that killáM. tuberculosisádirectly, we hoped to develop compounds that could render the bacterium susceptible to the immune system. We were successful in designing compounds that make laboratory-grown bacteria sensitive to a chemical produced during the immune response." Jason adds.
What the Researchers Tried to do
The team's strategy was to inhibit an enzyme found ináM. tuberculosisácalled the 20S proteasome.
The function of this enzyme is to dispose of damaged proteins within the bacterial cell, especially the proteins damaged by nitric oxide. The ability of the 20S proteasome to dispose of nitric oxide-damaged proteins helps the bacteria survive within the host.
Nitric oxide is a chemical produced by the innate immune system to help fight pathogens.
With the proteasome inhibited, proteins damaged by nitric oxide will accumulate inside the bacteria and cause their death.
But one key problem was that humans have a very similar system for the degradation of damaged proteins, and inhibition of this system could be lethal to cells.
So the researchers had to develop a compound that would selectively disrupt that bacterial proteasome, without significantly affecting the human version.
Inspiration from Nature
A bacterium calledáPseudomonas syringae, that infects plants, is known to produce compounds called syringolins that inhibit the plant proteasome.
These compounds are also known to inhibit the human proteasome and hold promise as anticancer agents.
Research had indicated that syringolins bound and inhibited the human proteasome degraded proteins with a specific chemical residue (valine) at two key positions.
Research had also indicated that the bacterial proteasome prefers to degrade proteins having two different chemical residues (tryptophan and glycine) at the same two key positions.
So, the researchers predicted that the compound would selectively inhibit the bacterial proteasome, if the syringolin analog in which the valine residue was swapped for structures resembling the tryptophan and glycine,
The researchers then designed and assessed the capacities of the compounds to inhibit both the human and bacterial proteasomes in a test tube.
Natural syringolin product was 160-fold more specific for the human proteasome, whereas the engineered syringolin analogs, in contrast, was 74-fold more specific for the bacterial proteasome.
"Using this rational design approach and chemical synthesis, we were able to generate selective inhibitors of theáM. tuberculosisá20S proteaseome," Sello said.
"In the best case, our engineering of the syringolins increased the inhibition of the bacterial enzyme by 220-fold, yet reduced the reaction with the human enzyme by 99.6%. Our success validated both the apparent substrate specificity of theáM. tuberculosisáproteasome and the structural model for proteasome inhibition by the syringolins."Sello added.
Susceptibility of the New Compound to Nitric Oxide
When the engineered syringolins was added to cultures ofáM. tuberculosisáin the presence and absence of a source of nitric oxide, they found that the bacteria treated with the compounds were highly susceptible to nitric oxide, whereas it did not inhibit the growth of human cell models.
"We were pleased to have engineered out the toxicity of the syringolins to human cells," Sello said. This means that an engineered syringolin is safe in humans."
"We've only modified the syringolins in two ways," Sello said. "There are many other possibilities for structural modification that could improve potency and other pharmacological properties of the molecules. We can now see a long but feasible pathway towards the development of a novel therapeutic agent for tuberculosis."
"One of the things that's clear in the treatment of tuberculosis is that combining drugs can be effective," he said. "So combining a blocker of the bacterium's defense against the immune system with a traditional antibiotic could be kind of a one-two punch."
This drug strategy could also be used alongside traditional antibiotics.
Kyle Totaro, Ph.D., who recently earned his Ph.D. from Brown, led Sello's team and the paper is published in the journaláACS Infectious Diseases
- Kyle A.Totaro et al. Rational Design of Selective and Bioactive Inhibitors of the Mycobacterium tuberculosis Proteasome. ACS Infectious Diseases; (2016) DOI: 10.1021/acsinfecdis.6b00172