Resisting resistance: Exposing sulfa drug mechanism

70 years of resistance

X-ray diffraction has been used to pin down the mode of action of the sulfa antibiotics, which were first used 70 years ago. The work could provide clues to developing a new generation of antibiotics that would have fewer side effects and could stave off bacterial drug resistance at least temporarily.

Researchers at St. Jude Children's Research Hospital in Memphis, Tennessee, have determined the structure of the bacterial target enzyme of sulfa drugs, dihydropteroate synthase (DHPS). Most pathogenic microbes require DHPS to help them synthesize folate, which is in turn used in the production of DNA and various essential amino acids. Stephen White, Richard Lee, Donald Bashford, Mi-Kyung Yun, Yinan Wu, Zhenmei Li, Ying Zhao, Brett Waddell and Antonio Ferreira worked with enzymes from gram-negative and gram-positive bacteria. They have used a variety of techniques to determine for the first time the key intermediate structure that is formed by DHPS in the advanced steps of folate production.

Importantly, from the perspective of antibiotic development, the structure they obtained helps to explain at the molecular level how sulfa drugs function and how resistance causing mutations help bacteria withstand them. The team says that the discovery could mark a major advance in both microbial biochemistry and anti-microbial drug discovery.

"The structure we found was totally unexpected and really opens the door for us and others to design a new class of inhibitors targeting DHPS that will help us avoid side effects and other problems associated with sulfa drugs," explains White. Colleague Richard Lee adds that: "Now we want to leverage this information to develop drugs against the opportunistic infections that threaten so many patients."

Sulfa drugs from the 1930s

Sulfa drugs were discovered in the 1930s and became the first antibiotics in widespread use. Although bacterial resistance emerged almost immediately the drugs were first used, an inevitable consequence of the use of any antibiotic given the nature of evolution, they are still widely used against emerging infectious diseases and to prevent infections in patients whose immune system is compromised, such as those undergoing cancer treatment or post-operative organ transplant patients.

Earlier studies showed that sulfa drugs target the enzyme DHPS and by emulating the structure of p-aminobenzoic acid, pABA block its activity. Given that DHPS advances folate production by accelerating the fusion of pABA and dihydropteridine pyrophosphate (DHPP) this ultimately has the effect of halting bacterial growth. However, until now researchers were unsure as to precisely how the disruption of the DHPS reaction took place and is disrupted by sulfa drugs.

The team used enzymes from gram-positive Bacillus anthracis and gram-negative Yersinia pestis, the bacteria that cause anthrax and plague, respectively. They first used computational techniques to predict the enzyme's activity and then XRD to capture the chemical reaction as it unfolds. They found that DHPP binds to a specific pocket in the enzyme and with the aid of a magnesium ion the binding process leads to the breakdown of DHPP and the release of a pyrophosphate ion.

The team explains that two long flexible loops create an intermediate structure that then allows pABA to enter and bind in a second short-lived pocket which gives pABA the opportunity to fuse with the cleaved DHPP. The XRD revealed not only the details of these chemical "actors" but identified for the first time the intermediate DHPP molecule in the cleaved state.

Critically, the results showed that chemical reaction takes place via an S_N1 (substitution nucleophilic, type 1) mechanism rather than the type 2,S_N2, as was expected. "This is a key finding for drug discovery because it reveals chemical features of the DHPS enzyme's active site that we can exploit in developing new drugs," explains tem member Donald Bashford.

The same XRD results also shed light on how bacteria develop drug resistance to the sulfa antibiotics. The team demonstrated that the binding sites of pABA and the sulfa drugs overlap, but that the sulfa drugs extend beyond the pocket in which pABA binds. When mutations emerge in the genes coding for this region those that distort the pocket so that pABA might still bind but sulfa can no longer mimic pABA as it did, then the bacteria with those mutations will be successful whereas those without will die. However, the research not only reveals how sulfa resistance can emerge but points to the transitory nature of the structure made by the two DHPS loops and so suggests a potential new target for novel antibiotics that would require several more mutations to emerge in concert for bacterial resistance to evolve against such drugs.

"When we set out on this project eight years ago, a goal was to truly understand the catalytic mechanism of the DHPS protein and how the inhibitors targeting it work. I am ecstatic we've succeeded," Lee enthuses. The researchers add that the plague enzyme turned out to be very well suited to the study because the two extended loops of its enzyme are free to form the short-lived structure and the pABA pocket when the enzyme is immobilized in the crystal offering a much clearer picture of the molecular circumstances.