Dual Inhibition of Mycobacterium tuberculosis and the Host TGFBR1 by an Anilinoquinazoline

Abstract

Tuberculosis (TB) control is complicated by the emergence of drug resistance. Promising strategies to prevent drug resistance are the targeting of nonreplicating, drug-tolerant bacterial populations and targeting of the host, but inhibitors and targets for either are still rare. In a cell-based screen of ATP-competitive inhibitors, we identified compounds with in vitro activity against replicating Mycobacterium tuberculosis (Mtb), and an anilinoquinazoline (AQA) that also had potent activity against nonreplicating and persistent Mtb. AQA was originally developed to inhibit human transforming growth factor receptor 1 (TGFBR1), a host kinase that is predicted to have host-adverse effects during Mtb infection. The structure-activity relationship of this dually active compound identified the pyridyl-6-methyl group as being required for potent Mtb inhibition but a liability for P450 metabolism. Pyrrolopyrimidine (43) emerged as the optimal compound that balanced micromolar inhibition of nonreplicating Mtb and TGFBR1 while also demonstrating improved metabolic stability and pharmacokinetic profiles.

Figure 1.

Overview of the Mtb whole-cell screen. A total of 879 compounds from the PKIS 1 and 2 collections were screened against an Mtb H37Rv strain constitutively expressing GFP. Percent inhibition was calculated relative to vehicle only on each plate. We defined hits as compounds showing more than 90% inhibition of GFP fluorescence compared to vehicle only. Twenty-six compounds showed above 50% increased fluorescence compared to vector only and were collapsed onto the x-axis. Compounds from PKIS 1 and 2 are separated by the vertical line.

Figure 2.

Structures, chemotypes, and MICs of hits. Structures of hit compounds with >90% inhibition of growth at 20 μM and their MIC₅₀, MIC₉₀, and MIC_90NR, the MIC₉₀ under hypoxic, nonreplicating (NR) conditions. The unit for all MICs is μM. ND: not determined.

Figure 3.

Activity of AQA against persisters. (A) Colony forming units after treatment of Mtb with 100× the MIC of INH and RIF in the absence and presence of 2xMIC_90NR of AQA. (B) Unlike AQA, ethambutol at 1xMIC in addition to INH and RIF does not further reduce cfu. One of three representative experiments shown, see Figure S2 for replicate experiments.

Figure 4.

Key H-bond interactions of AQA with the ATP-binding pocket of TGFBR1 from analysis of the crystal structure PDB: 3HMM.

Figure 5.

(A) Proposed modification of the AQA chemotype at rings A–C to develop SAR for Mtb inhibition, TGFBR1 inhibition, and metabolic stability. (B) The optimal pharmacological tool would lie in the overlap of the three SARs.

Figure 6.

Predicted metabolic hot spots determined by XenoSite.

Figure 7.

Mouse pharmacokinetic profile of indoloquinazoline 43.

Scheme 1.. Four-Step Synthesis of AQA Analogues^a

^aReagents and conditions: (i) Ar¹–CO₂H, HOBT, EDC, Et₃N; (ii) NaOH, EtOH; (iii) POCl₃, toluene; (iv) Ar²–NH₂, Bu^tONa, Pd₂(dba)₃, BINAP. (v) Ar²–OH, DIPEA, PrⁱOH.

Scheme 2.. Two-Step Synthesis of AQA Analogues^a

^aReagents and conditions: (i) 4-aminopyridine, NaH, DMF; (ii) Ar¹–B(OH)₂, Pd(dppf)Cl₂, K₂CO₃; (iii) Ar¹–SnBu₃, Pd(PPh₃)₄.