NITD-916

There is an unmet medical need for effective treatments against Mycobacterium abscessus pulmonary infections, to which cystic fibrosis (CF) patients are particularly vulnerable. Recent studies showed that the antitubercular drug isoniazid is inactive against M. abscessus due to the incapacity of the catalase-peroxidase to convert the pro-drug into a reactive metabolite that inhibits the enoyl-ACP reductase InhA. To validate InhA_MAB as a druggable target in M. abscessus, we assayed the activity of NITD-916, a 4-hydroxy-2-pyridone lead candidate initially described as a direct inhibitor of InhA that bypasses KatG bioactivation in Mycobacterium tuberculosis. The compound displayed low MIC values against rough and smooth clinical isolates in vitro and significantly reduced the bacterial burden inside human macrophages. Moreover, treatment with NITD-916 reduced the number and size of intracellular mycobacterial cords, regarded as markers of the severity of the infection. Importantly, NITD-916 significantly lowered the M. abscessus burden in CF-derived lung airway organoids. From a mechanistic perspective, NITD-916 abrogated de novo synthesis of mycolic acids and NITD-916-resistant spontaneous mutants harbored point mutations in InhA_MAB at residue 96. That NITD-916 targets InhA_MAB directly without activation requirements was confirmed genetically and by resolving the crystal structure of the protein in complex with NADH and NITD-916. These findings collectively indicate that InhA_MAB is an attractive target to be exploited for future chemotherapeutic developments against this difficult-to-treat mycobacterium and highlight the potential of NITD-916 derivatives for further evaluation in preclinical settings.

Purinergic and glutamatergic signalling pathways play a key role in regulating the activity of hypothalamic magnocellular neurosecretory neurons (MNNs).However, the precise cellular mechanisms by which ATP and glutamate act in concert to regulate osmotically driven MNN neuronal excitability remains unknown.Here, we report that ATP acts on purinergic P2 receptors in MNNs to potentiate in a Ca2+‐dependent manner extrasynaptic NMDAR function.The P2‐NMDAR coupling is engaged in response to an acute hyperosmotic stimulation, contributing to osmotically driven firing activity in MNNs.These results help us to better understand the precise mechanisms contributing to the osmotic regulation of firing activity and hormone release from MNNs.

The firing activity of hypothalamic magnocellular neurosecretory neurons (MNNs) located in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) is coordinated by the combined, fine‐tuned action of intrinsic membrane properties, synaptic and extrasynaptic signalling. Among these, purinergic and glutamatergic signalling pathways have been shown to play a key role regulating the activity of MNNs. However, the precise cellular mechanisms by which ATP and glutamate act in concert to regulate osmotically driven MNN neuronal excitability remains unknown. Whole‐cell patch‐clamp recordings obtained from MNNs showed that ATP (100 μM) induced an increase in firing rate, an effect that was blocked by either 4‐[[4‐formyl‐5‐hydroxy‐6‐methyl‐3‐[(phosphonooxy)methyl]2‐pyridinyl]azo]1,3‐benzenedisulfonic acid tetrasodium salt (PPADS) (10 μM) or kynurenic acid (1 mm). While ATP did not affect the frequency or magnitude of glutamatergic excitatory postsynaptic currents (EPSCs), it induced an inward shift in the holding current that was prevented by PPADS or kynurenic acid treatment, suggesting that ATP enhances a tonic extrasynaptic glutamatergic excitatory current. We observed that ATP‐potentiated glutamatergic receptor‐mediated currents were evoked by focal application of L‐glu (1 mm) and NMDA (50 μM), but not AMPA (50 μM). ATP potentiation of NMDA‐evoked currents was blocked by PPADS (10 μM) and by chelation of intracellular Ca2+ with BAPTA (10 mm). Finally, we report that a hyperosmotic stimulus (mannitol 1%, +55 mOsm/kgH2O) potentiated NMDA‐evoked currents and increased MNN firing activity, effects that were blocked by PPADS. Taken together, our data support a functional excitatory coupling between P2 and extrasynaptic NMDA receptors in MNNs, which is engaged in response to an acute hyperosmotic stimulus.