The program also includes novel antibacterial agents against drug-resistant tuberculosis, MRSA, VRE and other pathogens. Despite extensive research in the last 40 years, the drugs used to treat these infections are still limited to rather classical antibacterial drugs that target cell wall biosynthesis, nucleic acid synthesis, protein synthesis, etc. Furthermore, the widespread misuse of drugs and poor patient compliance have allowed bacteria to become resistant to drugs by means of their adaptive genetic machinery, leading to multi-drug resistant (MDR) strains of bacteria. Widespread bacterial resistance to existing therapeutics for bacterial infections mentioned above has been a key hurdle for the complete treatment. Therefore, in order to counter attack the adaptive genetic machinery of bacteria, there is a dire need for the identification of novel therapeutic targets. In this context, filamentous temperature sensitive protein Z (FtsZ), an essential bacterial cytokinesis protein, is a highly promising therapeutic target since the disruption of cell division would lead to the inhibition/arrest of bacterial infection. The Ojima Laboratory has discovered novel taxanes and benzimidazoles exhibit high potency against drug-sensitive and drug-resistant tuberculosis through efficient inhibition of FtsZ polymerization, which is crucial for bacterial cell division. The drug discovery efforts have been supported by design and synthesis of novel libraries of compounds and moderately high throughput (HTP) screening as well as rational optimization of the hit compounds.
Design and synthesis of novel anti-tubercular agents targeting FtsZ
Taxanes: Known tubulin inhibitors provide a good starting point
As FtsZ and tubulin have extensive structural and functional homology, a library of tubulin targeting taxanes, designed in our lab was screened to identify compounds which specifically target FtsZ. Several c-seco taxanes were found to exhibit MIC values of 1.25 - 2.5 uM with limited cytotoxicity.
SB-RA-5001 and its congeners displayed promising anti-tubercular activity against both drug sensitive and drug resistant strains. The scanning electron microscopy images of Micobacterium tuberculosis (Mtb) cells treated with SB-RA-20018 and SB-RA-5001 (Figure 1) show substantial elongation and filamentation, a phenotypic response to FtsZ inactivation
Figure 1: Electron micrograph of Mycobacterium tuberculosis cells. (A) Control, (B) SB‑RA-20018 and (C) SB-RA-5001.
Light scattering assay on Bacillus subtilis FtsZ (BsFtsZ)
Light scattering assay was carried out in order to check if SB-RA-2001 promoted FtsZ assembly (Figure 2). When the assay was carried out in the absence and presence or different concentrations of SB-RA-2001, it clearly displayed SB-RA-2001 promoted the assembly and bundling of BsFtsZ protofilaments.
Figure 2: Assembly of BsFtsZ in the absence (▲) and presence of 20 (□), 40 (○), and 60 μM (◆) SB-RA-2001 monitored by light scattering.
GTPase assay on BsFtsZ
GTPase activity plays an important role in FtsZ assembly. The effect of SB-RA-2001 on the GTP hydrolysis rate of FtsZ assembly was examined (Figure 3). SB-RA-2001 suppressed the GTPase activity of FtsZ in a concentration dependent manner.
Figure 3: The effects of SB-RA-2001 on the GTPase activity of BsFtsZ without (■) and with 0.01% (v/v) Triton X-100 (○) were determined after assembly for 5 min.
Analysis of binding of SB-RA-2001 on BsFtsZ
In silico analysis of SB-RA-2001 bound to FtsZ showed that the it binds in the interdomain cleft region,
adjacent to helix H7 of BsFtsZ (Figure 4).
Figure 4: (A) The putative binding site of SB-RA-2001 on FtsZ is localized between helix H7 and the C-terminus of FtsZ monomer. (B) Magnified view of the SB-RA-2001 binding cavity. (C) Interaction of SB-RA-2001 with the surrounding amino acid residues of FtsZ within 4 Å.
Singh, D.; Bhattacharya, A.; Rai, A.; Pal Singh Dhaked, H.; Awasthi, D.; Ojima, I.; Panda, D. Biochemistry. 2014, 53, 2979-2992.
Novel benzimidazoles targeting FtsZ as anti-tubercular agents
Based on the similarity of the benzimidazole moiety to the pyridopyrazine and pteridine pharamacophores identified by White, Reynolds and others as FtsZ inhibitors, we hypothesized that the benzimidazole framework might be a promising starting point for the development of novel FtsZ inhibitors (Figure 5).
Figure 5: Optimization of trisubstituted benzimidazoles
Optimization of 6-position
Initially trisubstituted benzimidazoles with diethyl amino substitution at the 6-position were synthesized. Further SAR studies led to substitution of the 6-position with pyrollidino group, which showed improved anti-tubercular activity compared to the diethyl amino substitution. Currently dimethyl amino group at the 6-position shows the best activity compared to other dialkyl amino groups (Figure 6).
Figure 6: SAR of the 6-positon of 2,5,6-trisubstituted benzimidazoles.
Mtb-FtsZ Polymerization inhibitory assay
Light scattering assay was performed in order to confirm the target was Mtb FtsZ. The extent of light scattering is directly proportional to the extent of FtsZ polymerization/aggregation. SB-P17G-C2 significantly reduced the light scattering demonstrating its inhibitory effect on the assembly of Mtb FtsZ (Figure 7).
Figure 7: Dose dependent inhibition of Mtb FtsZ polymerization by SB-P17G-C2
Antibacterial activity under hypoxic condition (LORA)
Table 1: LORA assayof benzimidazoles
SB-P3G2 and SB-P8B2 when tested against non-replicating Mtb bacteria under low oxygen conditions showed promising results. SB-P8B2 andSB-P3G2 at 4 mg/mL reduced the survival of M. tuberculosis H37Rv by 59% and 72%, respectively (Table 1).
In vivo efficacy of SB-P3G2 and SB-P17G-A20
SB-P3G2 reduced the bacterial load of M. tuberculosis H37Rv in lung and spleen of immune competent GKO mice and C57BL/6 mice (Figure 8).
Figure 8: (A) Scatter plots of the bacterial CFU counts from lungs (B) and spleen (:) from untreated control animals and the lungs (-) and spleen (;) of infected mice after treatment with SB-P3G2 delivered IP at 150 mg/kg for 9 consecutive days to immune incompetent GKO mice. SB-P3G2 reduced the bacterial load of M. tuberculosis H37Rv 0.71 _ 0.17 log10 CFU in the lungs and 0.41 _ 0.36 log10 CFU in spleen. (B) Scatter plots of the bacterial CFU counts from spleen (:) from untreated control animals and the spleen (;) of infected mice after treatment with SB-P3G2 delivered IP at 100 mg/kg BID for 10 consecutive days to immune competent C57BL/6mice. SB-P3G2 reduced the bacterial load of M. tuberculosis Erdman in the spleen log10 1.6 _ 0.49. In both studies, no outliers were identified by the Grubbs’ Test.
SB-P17G-A20 reduced the bacterial load in the lungs and spleen by 1.73 ± 0.24 Log10 CFU and 2.68 ± Log10 CFU respectively. SB-TB-1 administered via IP route significantly reduced the CFU in spleen and the results were comparable to INH. SB-TB-2 and SB-TB-3 reduced the bacterial load in the lungs by 6 and 5.4 Log10 CFU respectively, when given orally. In spleen, SB-TB-2 and SB-TB-3 reduced the bacterial load by 6.2 Log10 CFU , eliminating all living bacteria (Figure 9).
Figure 9: SB-P17G-A20 dosed IP at 50 mg/kg twice daily in the murine model of TB infection
In vitro and in vivo efficacy of the next generation lead compounds
The lead compounds SB-P17G-A33, SB-P17G-A38 and SB-P17GA42 displayed enhanced potency against M. tuberculosis H37Rv with MICs of 0.39 ± 0.16 μg/mL, 0.31 ± 0.22 μg/mL and 0.18 ± 0.1 μg/mL, respectively. These compounds displayed sigmoidal inhibition curves, is considered to be an important characteristic of an in vitro-in vivo relationship (Figure 10).
Figure 10: Dose response curves of SB-P17G-A33, SB-P17G-A38 and SB-P17GA42.
Killing characteristics of SB-P17G-A33, SB-P17G-A38 and SB-P17G-A42 against whole bacteria
Time dose curves were generated in order to identify the kill charecteristics of compounds against M. tuberculosis. The growth curve of M. tuberculosis in the presence of each of the compounds at different concentrations showed that all three compounds are concentration dependent inhibitors (Figure 11). SB-P17G-A38 reduced the number of bacteria by 2.8 Log10 CFU at 1X MIC by day 2 and by 2.9 Log10 CFU at day 6 (Figure 11a-c). SB-P17G-A42 reduced the number of viable bacteria by 2.5 Log10 CFU at day 2 at 1X MIC and continued to reduce the number of viable bacteria through day 6 by almost 4 Log10 CFU (Figure 11f). SB-P17G-A33 demonstrated less bactericidal activity reducing the bacterial load only 0.8-1.7 Log10 CFU during the first 2 days when dosed at MIC levels (Figure 11d). In addition, SB-P17G-A38 required 3-6X MIC to reduce the bacterial viability by 3.6-4.4 Log10 CFU, which is an equivalent level of reduction as to 1X of the other two lead compounds (Figure 11e).
Figure 11: Killing characteristics of SB-P17G-A33, SB-P17G-A38 and SB-P17G-A42 against whole Mtb bacteria
In vivoefficacy of SB-P17G-A33, SB-P17G-A38 and SB-P17G-A42 in the murine model of M.tuberculosisinfection when delivered IP or PO b.i.d.
SB-P17G-A33, SBP17G-A38 and SB-P17G-A42 were assessed in a rapid acute murine model of M.tuberculosis infection. SB-P17G-A38 reduced the bacterial load in the lungs by Log10 6.1 CFU when delivered PO and reduced the load in the lungs by Log10 6.3 CFU when delivered IP. SB-P17G-A42 reduced the bacterial load in the lungs by 5.6 Log10 CFU when delivered PO and reduced the load in the lungs by 5.8 Log10 CFU when delivered IP. SB-P17G-A38 and SB-P17GA42 displayed efficacy comparative to the front-line drug Isoniazid (Figure 10). SB-P17G-
A33 resulted in 1.7 and 2.5 Log10 CFU reduction in bacterial load in the lungs and spleen (Table 2)
Figure 12: In vivo efficacy of SB-P17G-A38 and SB-P17G-A42 dosed a) IP b) PO
Table 2: In vivo efficacy of SB-P17G-A33, SB-P17G-A38 and SB-P17G-A42 in the murine model of M.tuberculosis infection when delivered IP or PO b.i.d.
Inhibitors against other pathogens
As FtsZ is highly conserved in a range of bacterial strains, we screened our benzimidazole library against other bacterial strans such as F. tularensis, Y. pestis, B. thailandensis etc. as well and identified several lead compounds with MIC values in the range of 1 - 5 ug / mL (Figure 11).
Figure 13: In a screening against F. tularensis using 2,5,6- and 2,5,7-trisubstituted benzimidazoles, 21 compounds were found to inhibit bacterial cell growth by greater than 90%, with MIC90 ranging from 0.35-48.6 µg/mL.
Kumar, K.; Awasthi, D.; Lee, S.; Zanardi, I.; Ruzsicska, B.; Knudson, S.; Tonge, P.J.; Slayden, R.A.; Ojima, I. J. Med. Chem. 2010, 54, 374-381.
Awasthi, D.; Kumar, K.; Knudson, S.; Slayden, R.A.; Ojima, I.; J. Med. Chem. 2013, 56, 9756-9770.
Divya Awasthi, Kunal Kumar, Susan E. Knudson, Richard A. Slayden, and Iwao Ojima, J. Med. Chem. 2013, 56, 9756−9770
Kumar, K.; Awasthi, D.; Lee, S.; Cummings, J.E.; Knudson, S.E.; Slayden, R.A.; Ojima, I. Bioorg. Med. Chem. 2013, 21, 3318-3326.
Susan E. Knudson, Divya Awasthi, Kunal Kumar, Alexandra Carreau, Laurent Goullieux, Sophie Lagrange , He´ le`n Vermet, Iwao Ojima, Richard A. Slayden, Plos One, 2014, Volume 9, Issue 4, e93953.
Susan E. Knudson, Kunal Kumar, Divya Awasthi, Iwao Ojima, Richard A. Slayden, Tuberculosis ,94, 2014, 271- 276.
Iwao Ojima, Kunal Kumar, Divya Awasthi, Jacob G. Vineberg, Bioorg. Med. Chem. 22 (2014) 5060–5077
Susan E. Knudson, Divya Awasthi, Kunal Kumar, Alexandra Carreau, Laurent Goullieux, Sophie Lagrange, Hélène Vermet, Iwao Ojima, and Richard A. Slayden, JAC.
- Several lead benzimidazoles inhibited FtsZ polymerizaiton in a dose dependent manner.
- These compounds enhanced the GTPase activity by 3 - 4 folds.
- Scanning electron microscopy (SEM) images of Mtb cells treated with SB-P3G2 showed an absence of septum formation and slight cell elongation, indicative of FtsZ assembly inhibition.
- The drastic reduction in the mass of FtsZ polymer and bundling of FtsZ protofilaments was observed by transmission electrong microscopy (TEM) analysis of Mtb FtsZ treated with SB-P3G2.
On-going Research Efforts
- Extensive optimization of taxane-based and benzimidazole-based leads and screening of these libraries against other bacterial strains of interest.
- Focus on understanding the binding site of lead compounds:
- Photoaffinity labeling of FtsZ
- Co-crystallization of FtsZ with lead compounds
- In-silico docking
- ADMET and SAR study of lead compounds
- In-vivo assays to evaluate the metabolic and plasma stability of lead compounds