Toward Identification and Evaluation of Multitarget Inhibitors: Targeting Shikimate Pathway Enzymes in Mycobacterium tuberculosis

Abstract

Tuberculosis (TB) remains a significant global health issue. This ancient disease is further complicated by the rise of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains of Mycobacterium tuberculosis (MTB), making TB control increasingly challenging. Traditional single-target therapies have become less effective as MTB adapts and develops resistance mechanisms. This dissertation addresses this challenge by exploring multitarget drug development focused on the shikimate pathway—an essential metabolic route in MTB that is absent in humans and provides a therapeutic window for selective targeting.

The study focused on two key enzymes in the shikimate pathway: Mycobacterium tuberculosis shikimate kinase (MtSK) and shikimate dehydrogenase (MtSDH). A multi-stage approach, combining computational and experimental methodologies, was employed to identify multitarget inhibitors. Both MtSK and MtSDH were successfully expressed, purified, and characterized.

For MtSK, rates of catalysis as a function of ATP concentration revealed a kcat of 64 ± 2 s⁻¹ and a kon of (3.8 ± 0.1) × 10⁵ M⁻¹s⁻¹. Rates of catalysis as a function of shikimate concentration showed a kcat of 63 ± 1 s⁻¹ and a kon of (1.2 ± 0.3) × 10⁵ M⁻¹s⁻¹. Since MtSK does not naturally contain tryptophan residues, several tryptophan variants were constructed to explore intrinsic protein fluorescence, targeting key regions. For MtSK variants, rates of catalysis as a function of ATP concentration revealed the following kcat and kon values: D47W (kcat = 32 ± 1 s⁻¹, kon = (14 ± 1) × 10⁴ M⁻¹s⁻¹), F49W (kcat = 26 ± 1 s⁻¹, kon = (10 ± 2) × 10⁴ M⁻¹s⁻¹), E54W (kcat = 24 ± 2 s⁻¹, kon = (11 ± 1) × 10⁴ M⁻¹s⁻¹), V116W (kcat = 32 ± 1 s⁻¹, kon = (9 ± 1) × 10⁴ M⁻¹s⁻¹), and N151W (kcat = 35 ± 1 s⁻¹, kon = (11 ± 3) × 10⁴ M⁻¹s⁻¹). Rates of catalysis as a function of shikimate concentration revealed kcat and kon values as follows: D47W (kcat = 49 ± 1 s⁻¹, kon = (6.9 ± 0.3) × 10⁴ M⁻¹s⁻¹), F49W (kcat = 31 ± 1 s⁻¹, kon = (2.1 ± 0.1) × 10⁴ M⁻¹s⁻¹), E54W (kcat = 24 ± 2 s⁻¹, kon = (7 ± 1) × 10⁴ M⁻¹s⁻¹), V116W (kcat = 24 ± 1 s⁻¹, kon = (3 ± 1) × 10⁴ M⁻¹s⁻¹), and N151W (kcat = 33 ± 2 s⁻¹, kon = (6.3 ± 0.2) × 10⁴ M⁻¹s⁻¹).

For the MtSDH reverse reaction, rates of catalysis as a function of shikimate concentration revealed a kcat of 8.6 ± 0.3 s⁻¹ and a kon of (1.0 ± 0.1) × 10⁵ M⁻¹s⁻¹. Rates of catalysis as a function of NADP⁺ concentration yielded a kcat of 9.7 ± 0.1 s⁻¹ and a kon of (4.7 ± 0.7) × 10⁵ M⁻¹s⁻¹. In contrast, for the MtSDH forward reaction, rates of catalysis as a function of dehydroshikimate concentration showed a kcat of 18.5 ± 1.0 s⁻¹ and a kon of (4.3 ± 0.5) × 10⁵ M⁻¹s⁻¹. Rates of catalysis as a function of NADPH concentration yielded a kcat of 14.7 ± 0.4 s⁻¹ and a kon of (3.7 ± 0.2) × 10⁵ M⁻¹s⁻¹. The reverse reaction also revealed that NADP⁺ exhibited potent substrate-dependent inhibition. From the forward reaction, the inhibition constant (Ki) for NADP⁺ was calculated as 41 ± 4 µM, indicating that NADP⁺ competes with NADPH for the active site with a binding affinity comparable to that of NADPH.

To identify a set of compounds that may inhibit both enzymes, a library of 9,523 lead-like compounds was curated from the ZINC15 database. The compounds were filtered based on drug-likeness criteria, including Lipinski's Rule of Five, as well as toxicity profiles, and subsequently subjected to virtual screening using UCSF DOCK. Molecular docking studies focused on specific sub-pockets within the shikimate binding sites of both shikimate kinase (MtSK) and shikimate dehydrogenase (MtSDH), leading to the selection of the top 50 candidate compounds. These candidates underwent further refinement through ADME (absorption, distribution, metabolism, and excretion) and pharmacokinetic analyses to assess bioavailability, metabolic stability, and overall drug-like properties. Additionally, the ΔG binding of shikimate, calculated using the Normal Mode Entropy Approximation, was -17.31 ± 5.80 kcal/mol for MtSK and -26.28 ± 5.57 kcal/mol for MtSDH.

From the screening mentioned above, the top 16 compounds were further examined to map their molecular interactions with both enzymes, specifically evaluating hydrogen bonding and hydrophobic interactions with key active-site residues that are critical for binding stability and inhibitory potential. Out of those top 16 compounds, 11 were sourced from the National Cancer Institute (NCI) and proceeded to experimental validation.

Kinetic analyses revealed the inhibitory effects of several compounds, corroborating the in-silico predictions. Compound 16 emerged as the most potent inhibitor, demonstrating mixed noncompetitive inhibition against both MtSK and MtSDH. For MtSK, from the effect of [Compound 16] on Vmax, an apparent Ki of 25 ± 7 µM was obtained; and although the kapp vs. [Compound 16] did not permit a rigorous fit, we estimated that an apparent Ki < 35 µM would be necessary to account for the data. The affinity of Compound 16 for the MtSK-ATP and MtSK-ATP-SK complexes appears to be quite similar to one another. Similarly, against MtSDH, from the effect of [Compound 16] on Vmax, an apparent Ki of 38 ± 18 µM was obtained, and the effect of [Compound 16] on kapp produced an apparent Ki of 40 ± 18 µM. The data suggest that compound 16 interacts with comparable affinity to the MtSDH-NADP+ and MtSDH-NADP+-SK complexes. This dual inhibition not only disrupts two key enzymes in the shikimate pathway but also reduces the likelihood of resistance development, positioning Compound 16 as a highly viable lead candidate for further optimization.

Compound 01 exhibited mixed noncompetitive inhibition for MtSK and uncompetitive inhibition for MtSDH. An apparent Ki of 29 ± 12 µM was determined from the effect on Vmax, and an apparent Ki of 12.2 ± 8.5 µM from the effect on kapp. Our data indicate that Compound 01 binds to the MtSK-ATP and MtSK-ATP-shikimate complexes with a preference for the MtSK-ATP complex. For MtSDH, Compound 01 demonstrated uncompetitive inhibition with a Ki value exceeding 300 µM. Our data indicate that Compound 01’s primary interaction is with the MtSDH-NADP+-shikimate ternary complex. While the inhibitory capacity of Compound 01 is moderate, the clear inhibition patterns suggest the potential for structural refinement to enhance potency and specificity.

Compound 05 showed uncompetitive inhibition for both MtSK and MtSDH. For MtSK, Compound 05 appears to associate with the MtSK-ATP-shikimate ternary complex with an apparent Ki of 138 ± 61 µM. For MtSDH, our analysis indicated that the primary target for Compound 05 was also the MtSDH-NADP+-shikimate ternary complex. Although its potency is lower compared to Compounds 01 and 16, the distinct inhibition profile provides a foundation for structure-activity relationship (SAR) optimization aimed at improving its inhibitory capacity.

Compounds 03, 04, and 06 showed minimal inhibition of MtSDH within the concentration range evaluated in this study, and these same compounds showed no capacity to inhibit MtSK. The remaining compounds (02, 10, 11, 14, and 15) did not show a discernable ability to inhibit either MtSDH or MtSK. This is not to indicate that there is no future for these compounds in drug discovery efforts that may target the shikimate pathway. The kinetic data here represent preliminary findings that can be augmented by more in-depth studies in the future. In addition, some compounds could not be fully characterized due to experimental limitations. These will require further investigation, refined assay conditions, or improved compound stability.

The integrated in silico and in vitro approach offers a robust framework for multitarget drug discovery. While promising, the study acknowledges limitations, including replicating MTB’s intracellular environment and optimizing the pharmacokinetic properties of the inhibitors. In summary, this work advances antimicrobial drug development by identifying multitarget inhibitors against MTB, addressing the challenge of drug resistance, and moving us closer to a TB-free world.