Redirecting raltitrexed from cancer cell thymidylate synthase to Mycobacterium tuberculosis phosphopantetheinyl transferase.

There is a compelling need to find drugs active against Mycobacterium tuberculosis (Mtb). 4'-Phosphopantetheinyl transferase (PptT) is an essential enzyme in Mtb that has attracted interest as a potential drug target. We optimized a PptT assay, used it to screen 422,740 compounds, and identified raltitrexed, an antineoplastic antimetabolite, as the most potent PptT inhibitor yet reported. While trying unsuccessfully to improve raltitrexed's ability to kill Mtb and remove its ability to kill human cells, we learned three lessons that may help others developing antibiotics. First, binding of raltitrexed substantially changed the configuration of the PptT active site, complicating molecular modeling of analogs based on the unliganded crystal structure or the structure of cocrystals with inhibitors of another class. Second, minor changes in the raltitrexed molecule changed its target in Mtb from PptT to dihydrofolate reductase (DHFR). Third, the structure-activity relationship for over 800 raltitrexed analogs only became interpretable when we quantified and characterized the compounds' intrabacterial accumulation and transformation.

Mycobacterium tuberculosis PptT Inhibitors Based on Heterocyclic Replacements of Amidinoureas.

4'-Phosphopantetheinyl transferase (PptT) is an essential enzyme for Mycobacterium tuberculosis (Mtb) survival and virulence and therefore an attractive target for a tuberculosis therapeutic. In this work, two modeling-informed approaches toward the isosteric replacement of the amidinourea moiety present in the previously reported PptT inhibitor AU 8918 are reported. Although a designed 3,5-diamino imidazole unexpectedly adopted an undesired tautomeric form and was inactive, replacement of the amidinourea moiety afforded a series of active PptT inhibitors containing 2,6-diaminopyridine scaffolds.

In Vitro and In Vivo Inhibition of the Mycobacterium tuberculosis Phosphopantetheinyl Transferase PptT by Amidinoureas.

A newly validated target for tuberculosis treatment is phosphopantetheinyl transferase, an essential enzyme that plays a critical role in the biosynthesis of cellular lipids and virulence factors in Mycobacterium tuberculosis. The structure-activity relationships of a recently disclosed inhibitor, amidinourea (AU) 8918 (1), were explored, focusing on the biochemical potency, determination of whole-cell on-target activity for active compounds, and profiling of selective active congeners. These studies show that the AU moiety in AU 8918 is largely optimized and that potency enhancements are obtained in analogues containing a para-substituted aromatic ring. Preliminary data reveal that while some analogues, including 1, have demonstrated cardiotoxicity (e.g., changes in cardiomyocyte beat rate, amplitude, and peak width) and inhibit Cav1.2 and Nav1.5 ion channels (although not hERG channels), inhibition of the ion channels is largely diminished for some of the para-substituted analogues, such as 5k (p-benzamide) and 5n (p-phenylsulfonamide).

Characterization of Phosphopantetheinyl Hydrolase from Mycobacterium tuberculosis.

Phosphopantetheinyl hydrolase, PptH (Rv2795c), is a recently discovered enzyme from Mycobacterium tuberculosis that removes 4'-phosphopantetheine (Ppt) from holo-carrier proteins (CPs) and thereby opposes the action of phosphopantetheinyl transferases (PPTases). PptH is the first structurally characterized enzyme of the phosphopantetheinyl hydrolase family. However, conditions for optimal activity of PptH have not been defined, and only one substrate has been identified. Here, we provide biochemical characterization of PptH and demonstrate that the enzyme hydrolyzes Ppt in vitro from more than one M. tuberculosis holo-CP as well as holo-CPs from other organisms. PptH provided the only detectable activity in mycobacterial lysates that dephosphopantetheinylated acyl carrier protein M (AcpM), suggesting that PptH is the main Ppt hydrolase in M. tuberculosis. We could not detect a role for PptH in coenzyme A (CoA) salvage, and PptH was not required for virulence of M. tuberculosis during infection of mice. It remains to be determined why mycobacteria conserve a broadly acting phosphohydrolase that removes the Ppt prosthetic group from essential CPs. We speculate that the enzyme is critical for aspects of the life cycle of M. tuberculosis that are not routinely modeled. IMPORTANCE Tuberculosis (TB), caused by Mycobacterium tuberculosis, was the leading cause of death from an infectious disease before COVID, yet the in vivo essentiality and function of many of the protein-encoding genes expressed by M. tuberculosis are not known. We biochemically characterize M. tuberculosis's phosphopantetheinyl hydrolase, PptH, a protein unique to mycobacteria that removes an essential posttranslational modification on proteins involved in synthesis of lipids important for the bacterium's cell wall and virulence. We demonstrate that the enzyme has broad substrate specificity, but it does not appear to have a role in coenzyme A (CoA) salvage or virulence in a mouse model of TB.

Structural insights into phosphopantetheinyl hydrolase PptH from Mycobacterium tuberculosis.

The amidinourea 8918 was recently reported to inhibit the type II phosphopantetheinyl transferase (PPTase) of Mycobacterium tuberculosis (Mtb), PptT, a potential drug-target that activates synthases and synthetases involved in cell wall biosynthesis and secondary metabolism. Surprisingly, high-level resistance to 8918 occurred in Mtb harboring mutations within the gene adjacent to pptT, rv2795c, highlighting the role of the encoded protein as a potentiator of the bactericidal action of the amidinourea. Those studies revealed that Rv2795c (PptH) is a phosphopantetheinyl (PpT) hydrolase, possessing activity antagonistic with respect to PptT. We have solved the crystal structure of Mtb's phosphopantetheinyl hydrolase, making it the first phosphopantetheinyl (carrier protein) hydrolase structurally characterized. The 2.5 Å structure revealed the hydrolases' four-layer (α/ß/ß/α) sandwich fold featuring a Mn-Fe binuclear center within the active site. A structural similarity search confirmed that PptH most closely resembles previously characterized metallophosphoesterases (MPEs), particularly within the vicinity of the active site, suggesting that it may utilize a similar catalytic mechanism. In addition, analysis of the structure has allowed for the rationalization of the previously reported PptH mutations associated with 8918-resistance. Notably, differences in the sequences and predicted structural characteristics of the PpT hydrolases PptH of Mtb and E. coli's acyl carrier protein hydrolase (AcpH) indicate that the two enzymes evolved convergently and therefore are representative of two distinct PpT hydrolase families.

Opposing reactions in coenzyme A metabolism sensitize Mycobacterium tuberculosis to enzyme inhibition.

Mycobacterium tuberculosis (Mtb) is the leading infectious cause of death in humans. Synthesis of lipids critical for Mtb's cell wall and virulence depends on phosphopantetheinyl transferase (PptT), an enzyme that transfers 4'-phosphopantetheine (Ppt) from coenzyme A (CoA) to diverse acyl carrier proteins. We identified a compound that kills Mtb by binding and partially inhibiting PptT. Killing of Mtb by the compound is potentiated by another enzyme encoded in the same operon, Ppt hydrolase (PptH), that undoes the PptT reaction. Thus, loss-of-function mutants of PptH displayed antimicrobial resistance. Our PptT-inhibitor cocrystal structure may aid further development of antimycobacterial agents against this long-sought target. The opposing reactions of PptT and PptH uncover a regulatory pathway in CoA physiology.

Unlocking Endosomal Entrapment with Supercharged Arginine-Rich Peptides.

Endosomal entrapment is a common bottleneck in various macromolecular delivery approaches. Recently, the polycationic peptide dfTAT was identified as a reagent that induces the efficient leakage of late endosomes and, thereby, enhances the penetration of macromolecules into the cytosol of live human cells. To gain further insights into the features that lead to this activity, the role of peptide sequence was investigated. We establish that the leakage activity of dfTAT can be recapitulated by polyarginine analogs but not by polylysine counterparts. Efficiencies of peptide endocytic uptake increase linearly with the number of arginine residues present. In contrast, peptide cytosolic penetration displays a threshold behavior, indicating that a minimum number of arginines is required to induce endosomal escape. Increasing arginine content above this threshold further augments delivery efficiencies. Yet, it also leads to increasing the toxicity of the delivery agents. Together, these data reveal a relatively narrow arginine-content window for the design of optimally active endosomolytic agents.

An active role for the ribosome in determining the fate of oxidized mRNA.

Chemical damage to RNA affects its functional properties and thus may pose a significant hurdle to the translational apparatus; however, the effects of damaged mRNA on the speed and accuracy of the decoding process and their interplay with quality-control processes are not known. Here, we systematically explore the effects of oxidative damage on the decoding process using a well-defined bacterial in vitro translation system. We find that the oxidative lesion 8-oxoguanosine (8-oxoG) reduces the rate of peptide-bond formation by more than three orders of magnitude independent of its position within the codon. Interestingly, 8-oxoG had little effect on the fidelity of the selection process, suggesting that the modification stalls the translational machinery. Consistent with these findings, 8-oxoG mRNAs were observed to accumulate and associate with polyribosomes in yeast strains in which no-go decay is compromised. Our data provide compelling evidence that mRNA-surveillance mechanisms have evolved to cope with damaged mRNA.