# Target based discovery of next generation pyrazinamide

> **NIH NIH R01** · HACKENSACK UNIVERSITY MEDICAL CENTER · 2022 · $791,696

## Abstract

Tuberculosis (TB) is a main cause of death for people living with AIDS. In immune-competent populations, drug
therapy and immunity join forces to win the fight against Mycobacterium tuberculosis (Mtb). In HIV-positive
individuals on the other hand, chemotherapy must fully sterilize all infection sites. There is an urgent medical
need to develop more potent `sterilizing' drugs.
The inclusion of pyrazinamide (PZA) into the TB regimen allowed reduction of treatment time to six months, while
maintaining low relapse rates. The pharmacological basis for PZA's remarkable sterilizing activity in patients
remains obscure, considering the drug's poor in vitro potency (MIC = 30-100 µg/mL). In the rabbit model of active
TB, an animal model that recapitulates the various lung lesion types observed in human TB disease, we showed
that PZA not only penetrates caseous necrotic lesions but also sterilizes them. In an ex vivo assay using caseum
from infected rabbits, we showed that PZA kills non-growing, drug tolerant Mtb. These findings provide an
explanation for the clinically observed treatment shortening effect of PZA: the drug reaches difficult-to-penetrate
TB lesions and kills recalcitrant `persister' Mtb. However, consistent with the modest in vitro potency of PZA,
onset of lesion sterilization is slow and concentrations required to kill Mtb in ex vivo caseum are high. Based on
these findings, the logical way forward is to improve the potency of PZA while maintaining its unique lesion
penetration and sterilization properties. To enable the rational optimization of PZA, we identified aspartate
decarboxylase PanD, required for coenzymeA biosynthesis, as a the first genetically, biochemically and
biophysically validated target of PZA. Consistent with poor whole cell activities of PZA, affinity of the drug (more
specifically its bioactive component pyrazinoic acid, POA) for PanD was in the µM range, confirming room for
improvement. Interestingly, mechanism of action studies revealed a novel antibacterial on-target mechanism
whereby, rather than inhibiting PanD's catalytic activity, binding of POA to PanD appears to trigger degradation
of the protein by the caseinolytic protease ClpC1.
Here, we propose to build on our discoveries and 1. fully characterize the novel on-target mechanism by which
the drug induces degradation of its target, 2. exploit in vitro pharmacological `sterilizing' models and PanD-based
assays for the discovery of novel PZA analogs with improved potency and sterilizing activity, and 3. characterize
lesion specific growth and replication status of PZA resistant panD mutant Mtb vs. wild type Mtb in the TB rabbit
model with and without PZA treatment.
In summary, we have identified key pharmacological properties responsible for the treatment shortening activity
of PZA in clinical settings, as well as the molecular target of the drug, and we propose a `first-in-class to best-in-
class' program to exploit these findings and rationally design the `n...

## Key facts

- **NIH application ID:** 10404533
- **Project number:** 5R01AI106398-09
- **Recipient organization:** HACKENSACK UNIVERSITY MEDICAL CENTER
- **Principal Investigator:** Thomas Dick
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $791,696
- **Award type:** 5
- **Project period:** 2013-08-01 → 2023-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10404533

## Citation

> US National Institutes of Health, RePORTER application 10404533, Target based discovery of next generation pyrazinamide (5R01AI106398-09). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10404533. Licensed CC0.

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