Tuberculosis (TB) and its drug resistant forms kills more people than any other infectious disease. This fact emphasizes the need to identify new drugs to treat TB. 2-Aminothiophenes (2AT) have been reported to inhibit Pks13, a validated anti-TB drug target. We synthesized a library of 42 2AT compounds. Among these, compound 33 showed remarkable potency against Mycobacterium tuberculosis (Mtb) H37RV (MIC = 0.23 μM) and showed an impressive potency (MIC = 0.20-0.44 μM) against Mtb strains resistant to isoniazid, rifampicin and fluoroquinolones. The site of action for the compound 33 is presumed to be Pks13 or an earlier enzyme in the mycolic acid biosynthetic pathway. This inference is based on structural similarity of the compound 33 with known Pks13 inhibitors, which is corroborated by mycolic acid biosynthesis studies showing that the compound strongly inhibits the biosynthesis of all forms of mycolic acid in Mtb. In summary, these studies suggest 33 represents a promising anti-TB lead that exhibits activity well below toxicity to human monocytic cells.

Glycoside hydrolases (GH) are a large family of hydrolytic enzymes found in all domains of life. As such, they control a plethora of normal and pathogenic biological functions. Thus, understanding selective inhibition of GH enzymes at the atomic level can lead to the identification of new classes of therapeutics. In these studies, we identified a 4-⍺-glucoside of valienamine (8) as an inhibitor of Streptomyces coelicolor (Sco) GlgE1-V279S which belongs to the GH13 Carbohydrate Active EnZyme family. The results obtained from the dose–response experiments show that 8 at a concentration of 1000 µM reduced the enzyme activity of Sco GlgE1-V279S by 65%. The synthetic route to 8 and a closely related 4-⍺-glucoside of validamine (7) was achieved starting from readily available D-maltose. A key step in the synthesis was a chelation-controlled addition of vinylmagnesium bromide to a maltose-derived enone intermediate. X-ray structures of both 7 and 8 in complex with Sco GlgE1-V279S were solved to resolutions of 1.75 and 1.83 Å, respectively. Structural analysis revealed the valienamine derivative 8 binds the enzyme in an E2 conformation for the cyclohexene fragment. Also, the cyclohexene fragment shows a new hydrogen-bonding contact from the pseudo-diaxial C(3)–OH to the catalytic nucleophile Asp 394 at the enzyme active site. Asp 394, in fact, forms a bidentate interaction with both the C(3)–OH and C(7)-OH of the inhibitor. In contrast, compound 7 disrupts the catalytic sidechain interaction network of Sco GlgE1-V279S via steric interactions resulting in a conformation change in Asp 394. These findings will have implications for the design other aminocarbasugar-based GH13-inhibitors and will be useful for identifying more potent and selective inhibitors.

α,α′‐Trehalose plays roles in the synthesis of several cell wall components involved in pathogenic mycobacteria virulence. Its absence in mammalian biochemistry makes trehalose‐related biochemical processes potential targets for chemotherapy. The trehalose 6‐phosphate synthase (TPS)/trehalose 6‐phosphate phosphatase (TPP) pathway, also known as the OtsA/OtsB2 pathway, is the major pathway involved in the production of trehalose in Mycobacterium tuberculosis (Mtb). In addition, TPP is essential for Mtb survival. We describe the synthesis of α,α′‐trehalose derivatives in the forms of the 6‐phosphonic acid 4 (TMP), the 6‐methylenephosphonic acid 5 (TEP), and the 6‐N‐phosphonamide 6 (TNP). These non‐hydrolyzable substrate analogues of TPP were examined as inhibitors of Mtb, Mycobacterium lentiflavum (Mlt), and Mycobacterium triplex (Mtx) TPP. In all cases the compounds were most effective in inhibiting Mtx TPP, with TMP [IC50=(288±32) μm] acting most strongly, followed by TNP [IC50=(421±24) μm] and TEP [IC50=(1959±261) μm]. The results also indicate significant differences in the analogue binding profile when comparing Mtb TPP, Mlt TPP, and Mtx TPP homologues.

C7/C8-cyclitols and C7N-aminocyclitols find applications in the pharmaceutical sector as α-glucosidase inhibitors and in the agricultural sector as fungicides and insecticides. In this study, we identified C7/C8-cyclitols and C7N-aminocyclitols as potential inhibitors of Streptomyces coelicolor (Sco) GlgEI-V279S based on the docking scores. The protein and the ligand (targets 11, 12, and 13) were prepared, the states were generated at pH 7.0 ± 2.0, and the ligands were docked into the active sites of the receptor via Glide™. The synthetic route to these targets was similar to our previously reported route used to obtain 4-⍺-glucoside of valienamine (AGV), except the protecting group for target 12 was a p-bromobenzyl (PBB) ether to preserve the alkene upon deprotection. While compounds 11–13 did not inhibit Sco GlgEI-V279S at the concentrations evaluated, an X-ray crystal structure of the Sco GlgE1-V279S/13 complex was solved to a resolution of 2.73 Å. This structure allowed assessment differences and commonality with our previously reported inhibitors and was useful for identifying enzyme–compound interactions that may be important for future inhibitor development. The Asp 394 nucleophile formed a bidentate hydrogen bond interaction with the exocyclic oxygen atoms (C(3)-OH and C(7)-OH) similar to the observed interactions with the Sco GlgEI-V279S in a complex with AGV (PDB:7MGY). In addition, the data suggest replacing the cyclohexyl group with more isosteric and hydrogen bond–donating groups to increase binding interactions in the + 1 binding site.

No abstract

No abstract

In 2016, 8.1 % of new cases (601,000 people) and 20 % of previously treated cases of Tuberculosis (TB) were diagnosed as Multiply Drug Resistant Tuberculosis (MDR‐TB) cases, amongst which 10 % are Totally Drug Resistant Tuberculosis (TDR‐TB) cases worldwide. In America, MDR‐TB new cases make 1.5 % of the cases among which none of the TDR‐TB cases were successfully treated in 2016. In addition to TB and HIV treatment being contraindicated, the rise of MDR‐TB and TDR‐TB emphasize the need for new drugs and drug targets. Identified in 2010, the maltosyltransferase GlgE is an interesting drug target in that it has no homologues in humans and its inhibition results in the death of the microorganism in 14 days. Based on the structure of GlgE's natural ligand, maltose‐1‐phosphate, and the known enzymatic mechanism, substrate and transition‐state analogs were designed as potential inhibitors of the enzyme and co‐crystallized with an MtbGlgE homolog. To improve the resolution of our X‐ray structures to more accurately analyze the interactions between the inhibitors and the residues in the active site, ScoGlgEI has been used as a surrogate. Sharing 53 % identity with MtbGlgE, we created a ScoGlgEI‐V279S variant as to obtain 100 % identity between the active sites of the two homologues. Based on the information obtained from X‐ray structures of ScoGlgEI‐V279S in complex with our initial inhibitor, 2,5‐dideoxy‐3‐O‐α‐D‐glucopyranosyl‐2,5‐imino‐D‐mannitol (DDGIM), a 2‐fold inhibition improvement has been made on our second‐generation inhibitors bringing the IC50 from 102 ± 8 μM to 45 ± 4 μM. Recently published, the crystal structures of ScoGlgEI‐V279S in complex with these early‐ and late‐dissociative transition‐state‐like inhibitors were solved at 2.5 Å and 3.2 Å showing that improvement was obtained through the addition of a phosphonate to our DDGIM inhibitor, resulting in an increased number of hydrogen bonded interactions between the enzyme and the inhibitors. To increase potency of our next generation inhibitor, additional interactions with the enzyme were sought outside the active site. The release of a structure of ScoGlgEI‐V279S in complex with its substrate brought more information to increase the number of interactions between ScoGlgEI‐V279S and the next generation of inhibitors. The details of the crystal structure of ScoGlgEI‐V279S in complex with our third‐generation inhibitor was obtained at 3.35 Å and the determined inhibitory activity will be discussed.Support or Funding InformationNIH grant AI105084This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.