A medicinal chemists' guide to the unique difficulties of lead optimization for tuberculosis

Abstract

Tuberculosis is a bacterial disease that predominantly affects the lungs and results in extensive tissue pathology. This pathology contributes to the complexity of drug development as it presents discrete microenvironments within which the bacterium resides, often under conditions where replication is limited and intrinsic drug susceptibility is low. This consolidated pathology also results in impaired vascularization that limits access of potential lead molecules to the site of infection. Translating these considerations into a target-product profile to guide lead optimization programs involves implementing unique in vitro and in vivo assays to maximize the likelihood of developing clinically meaningful candidates.

Keywords: Drug discovery; Tuberculosis.

Figure 1

The traditional methodology for lead optimization is driven by the “three Ms”. Left represents determination of the Minimum Inhibitory Concentration (MIC) with the top panel showing a scanning electron micrograph of Mtb and the bottom showing a transmission electron micrograph of the same culture. The center panels show the second M, mouse efficacy is typically assessed as both single agents and as combinations of agents. The lower panel shows a Hematoxylin and Eosin (H&E) stain of murine TB lesions which are typically not well organized discrete structures but rather loose aggregates of infected cells. The right panels show the third M, man, with the top figure being a 3-dimensional reconstruction of Computed Tomography (CT) scans of a patient with extensive excess high-density lesions in the lungs. The bottom shows an H&E stain from a human lung resection surgery in which both acellular, caseous lesions (A) and necrotic lesions in the process of cavitation are present (B).

Figure 2

The development of cavitary TB in adult humans and the implications for drug discovery. Initial infection of pulmonary macrophages is followed by migration of these infected cells into the airway interstitium where they attract tissue macrophages and migrating monocytes and other immune cells to initiate formation of a cellular granuloma. Vascularization of this structure is initially intact but as it expands and becomes caseous vascularization is minimal. TB bacteria contained within this granuloma experience a variety of stresses resulting in them becoming drug tolerant while at the same time the vasculature becomes limiting. Eventually this structure breaks down and the bacteria, completely insulated from the immune system, are released into the airway (and the bloodstream) where they can be expelled and initiate another cycle of infection in a new host. Cavity formation is a hall mark of TB disease and the organisms in cavities are the most difficult to eradicate resulting in long durations of treatment.

Figure 3

(A) Typical well-circumscribed granuloma with central caseous necrosis devoid of vascular supply, surrounded by a layer of immune cells and a thick fibrotic rim; (B) Cavitary lesion showing softened caseum in the center (partially disintegrated due to the sectioning procedure). The cellular and fibrotic rim of the cavity is rich in blood capillaries (indicated by the white arrows), effectively bringing inflammatory cells and drugs to the cellular part of the lesion. Hematoxylin and eosin stain, 12.5x magnification.

Figure 4

MALDI mass spectrometry (MS) images showing moxifloxacin (MXF) distribution within rabbit granulomas (A), MXF in a human necrotic nodule (B), and PA-824 in a rabbit necrotic granuloma (C). On the lower left panel, an H&E stained reference tissue section is displayed below the corresponding MALDI MS image. White circles surround a necrotic granuloma. The necrotic center containing caseating material (black arrows) is visible as a light pink center in the H&E stained reference. (B) and (C): the white contour lines highlight the necrotic center of each granuloma, surrounded by the cellular region on the lower right side in each case. The poor distribution of MXF and PA-824 within caseum relative to the cellular cuff is clearly visible in all samples. Signal intensities are shown as a fixed scale.