Synergy graph illustrating the efficacy of the dual inhibition Mtb treatment strategy. (Credit: Campbell lab)
Tuberculosis is both curable and preventable, yet each year, it still kills more people than any other infectious disease. One reason is that current treatments hinge on rifampicin, an antibiotic that blocks bacterial transcription and forms the cornerstone of a multidrug regimen-rising drug resistance has revealed the limits of leaning so heavily on a single point of attack.
Now, a new study demonstrates one way to rethink that strategy-not by finding a new cornerstone, but by pairing rifampicin with a second inhibitor that strikes the same pathway from a different angle.
The findings, published in Nature Microbiology, show that rifampicin becomes more powerful when paired with a second compound, known as AAP-SO₂, and that these drugs can together not only suppress TB resistance but also target the dormant bacteria that standard drugs struggle to kill. The two compounds work by exploiting a weakness in a common resistance mutation: evading rifampicin comes at a cost to the bacteria, forcing it to transcribe genetic material more slowly-where a second drug turns their attempted resistance into a disadvantage.
The results provide a pathway for the development of future dual-inhibitor drug strategies against tuberculosis, and reframe TB treatment as a precision strategy that could be built around better understanding molecular bottlenecks in resistant strains. "Basic science is putting us one step ahead of bacteria," says Elizabeth Campbell, head of the Laboratory of Molecular Pathogenesis at Rockefeller. "Thanks to that kind of research into TB's transcription and genetics, we can now strategize how to prevent resistance, or even exploit resistance for the development of new therapies."
TB's new Achilles' heel
Tuberculosis treatment leans heavily on rifampicin, a frontline antibiotic that blocks the pathogen's RNA polymerase (RNAP), the enzyme that transcribes DNA into RNA. Rifampicin can also kill some of the inert bacteria lingering inside the clusters of compromised immune cells in the lung that act as reservoirs for TB. But rifampicin resistance is on the rise.
"As a South African scientist with clinical experience, I have witnessed how tuberculosis remains one of the most devastating health challenges for families and communities back home," says Vanisha Munsamy-Govender, a scientist in Jeremy Rock's Laboratory of Host-Pathogen Biology.
Resistance to rifampicin is driven largely by a common mutation in RNAP known as βS450L. Previous work from the Campbell and Rock labs demonstrated that one tradeoff of this resistance mutation is that it also causes RNAP to run more slowly during the latter stage of transcription, a process known as elongation, which makes it more likely that transcription as a whole will stall out. The team wondered whether this defect could be exploited. Since rifampicin targets only the early promoter escape step of transcription, it made sense that hitting the sluggish and error-prone elongation step in tandem might keep TB down for the count.
"Earlier work from the Campbell and Rock labs really laid out the vulnerability of these resistant strains," says Barbara Bosch, an instructor in clinical investigation who has worked in both laboratories. "So we started asking: how can we go from that knowledge to new combinations of drugs that better target bacteria, especially in those hard-to-reach clusters?"
Designing the one-two punch
The researchers began by testing whether using two drugs to block different steps of the same pathway-a strategy known as vertical inhibition-could outmaneuver resistant TB. To try it, they paired rifampicin with AAP-SO₂, a compound that was less a drug candidate than a proof-of-concept probe, to see whether dual inhibition could outperform rifampicin on its own.
After confirming that AAP-SO₂ binds directly to bacterial RNAP and specifically slows the elongation stage of transcription, the team found that it attaches at a different site than rifampicin. This provided molecular evidence that the two compounds should, in theory, act in concert and at the same time, each blocking a different step of the transcription pathway-a combination lethal to the bacteria.
"Cells have to transcribe genes to survive; they don't really have a way around it," Bosch says.
Their plan worked. AAP-SO₂ wiped out the rifampicin-resistant mutant βS450L, exploiting the slowed transcription that helped it dodge rifampicin but left it vulnerable to a second hit. The effect was so strong that this mutation was effectively driven out of the bacterial population as it was again rendered vulnerable to rifampicin. Even more striking results were seen when the researchers moved from liquid culture to a rabbit model designed to mimic dormant clusters. In culture, rifampicin and AAP-SO₂ behaved additively, each contributing its own effect without enhancing the other. But in cluster-like tissue, the drugs became synergistic, killing far more bacteria together than either drug alone. The findings suggest that the addition of this second compound increased the potency of rifampicin 30-fold.
"AAP-SO₂ slows the emergence of resistance and works synergistically with rifampicin to eliminate what makes TB so difficult to cure," says Munsamy-Govender.
Together, these results make a compelling case for reinforcing rifampicin rather than replacing it as the go-to treatment. Because AAP-SO₂ is not a drug candidate, the next steps involve building a stable derivative of the compound; the team has already filed a provisional patent on the dual-inhibition strategy described in their paper.
But the implications reach beyond one compound. As the researchers demonstrated, TB drug development could begin to shift toward a precision medicine approach, in which companion drugs are matched to the vulnerabilities of a particular strain-much as AAP-SO₂ was paired with rifampicin here to exploit a specific resistance mutation. As a result, resistance mutations, long viewed only as a threat, can now reveal new therapeutic footholds when studied closely, and robust mechanistic insight can now be converted into strategy.
"We've shown how basic science can guide therapeutic strategy," Rock says. "By deeply understanding, mechanistically, what happens when these bugs become resistant to antibiotics, we can start to rationally design ways of combatting that in the clinic."
