MRSA bacteria cause staph infections that are resistant to many antibiotics. These bacteria are among the
Current treatments for MRSA include a combination of antifolate drug trimethoprim (TMP) and an antibiotic known as sulfamethoxazole. These treatments
DHFR inhibitors work similarly to a lock and key. They bind to MRSA enzymes by fitting into a 3D structure that only allows molecules that fit precisely to enter.
However, over time, MRSA has mutated and become resistant to these drugs. One reason for this is the well-known F98Y mutation, which causes a slight change to the 98th amino acid on the DHFR enzyme and ultimately makes inhibitor drugs unable to bind to it like a lock and key.
For some time, scientists have been unable to map the mechanisms underlying how this mutation leads to drug resistance. However, they know that identifying how these mutations make MRSA resistant to treatment will help them predict new mutations and develop new drugs to target MRSA more effectively.
In a recent study, researchers at Duke University in Durham, NC, investigated how MRSA’s mutations confer resistance to certain treatments.
They found that MRSA’s resistance stems from the F98Y mutation causing a chirality preference for the NADPH co-factor, the catalyst for mutations in DHFR.
Chirality refers to the handedness of a molecule, whereby it is not possible to overlay its mirror image without overlaps — for example, in the same way that a person cannot overlay their left and right hand without their thumbs sticking out on different sides.
“This is the first example of an enzyme that exploits the chirality of its co-factor in order to evade its inhibitors,” Graham Holt, a grad student at the Donald Lab at Duke University, told Medical News Today. “Now that we see this happening, that will help inform computational strategies to develop better inhibitors.”
The research appears in the journal PLOS Computational Biology.
The researchers initially tried to predict MRSA’s F98Y mutations using Open Source Protein REdesign for You (OSPREY), a suite of programs for computational structure-based protein design. After a series of tests that did not yield results, they decided to reexamine MRSA’s starting structure.
They did so using
This map is similar to a cloud in an empty space, with the thicker and thinner areas revealing the location of the molecule’s atoms.
Using this electron density map, computational methods can predict what potential molecular and atomic structure of the protein would best fit within these contours.
The researchers used various programs to try to predict the molecular makeup of the NADPH protein that would fit into the cloud-like electron density map. They knew that the NADPH co-factor molecule was somewhere in the electron density data, so they asked the program to try for a best fit within the contours of the predefined space using a right-handed configuration.
However, as the program could not fit the NADPH co-factor molecule into the space in this right-handed configuration, it flipped it into a left-hand configuration, ultimately causing a better predicted fit.
The researchers did not realize that the program had “flipped” the molecule to improve the predicted fit. Unaware of the flipped chirality, the researchers proceeded to analyze the protein again with OSPREY.
“Protein design is very sensitive to all sorts of things like chirality,” lead author Bruce R. Donald, who is James B. Duke Distinguished Professor of Computer Science and Mathematics, Chemistry and Biochemistry, told MNT.
“When we tried to analyze the structure using protein design in order to see if resistance mutations could be predicted, problems came up. Then we noticed that the NADPH molecule was flipped!” he added.
This flipped chirality, say the authors, changed the protein’s structural basis for resistance. Much like in two-factor authentication, they found that the well-known single enzyme mutation led to the flipped co-factor binding site, contributing to MRSA’s increasing resistance.
“This unleashed a rather intense round of experimentation,” said Dr. Donald. “In the final examination, we found the co-factor was not only flipped, [but it was also] flipped and cyclized — which means it has an extra ring — from the expected model.”
“Once we were certain that the chemistry was understood, we then undertook more experiments and more computation to try to understand the possible biological and biochemical implications of this discovery,” he explained.
During these experiments and computational exercises, the researchers found that accounting for the flipped chirality made their results better match experimental measurements of inhibitor potency.
In response to a question on what may have caused the observed chiral evasion, Dr. Donald said: “The short answer is we don’t know. We believe that the resistance mutation F98Y remodels the co-factor binding site so that it prefers the flipped/cyclized version of NADPH. And it simultaneously changes the active site of the enzyme to [change how drugs can bind to it].”
“Let me break that down. Some drug molecules also have chiral centers — [an atom that has four unique atoms or groups attached to it]. In the [regular] version of the protein, drugs (in the class we studied) with either kind of chirality — [i.e., either mirror image of the protein] — bind tightly, and both inhibit the enzyme,” he clarified.
“But in enzymes with this resistance mutation, only the drugs with one particular ‘handedness’ can bind tightly! Drugs with the other handedness that previously worked are now rendered much less effective.”
– Dr. Bruce Donald
The researchers conclude that a switched NADPH chirality may contribute to MRSA’s resistance to drugs and that scientists should take this chiral evasion into account when designing new drugs for the bacteria.
They note that although their evidence is based on strong computational and biochemical evidence, in vivo experiments are necessary to determine whether these mechanisms really occur in living organisms and the extent of their importance.
Dr. Donald added: “Our analysis is basically about thermodynamics. But we think that an important component would be kinetics — the rate at which these reactions and changes occur. We plan to measure that in the future to provide a full answer.”
When MNT asked how these findings could lead to the development of better treatments for drug-resistant bacteria, Dr. Donald said:
“Most drug design is reactive, where we wait until resistance mutations emerge in a clinical setting before trying to combat them. When we discover new resistance mechanisms, as in this paper, the hope is that we can use that understanding to predict new resistance mutations that will emerge in the future and even counter them while drugs are still being developed in the laboratory.”