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Many bacterial pathogens, including Staphylococcus aureus, require inosine 5'-monophosphate dehydrogenase (IMPDH) for infection, making this enzyme a promising new target for antibiotics. Although potent selective inhibitors of bacterial IMPDHs have been reported, relatively few have displayed antibacterial activity. Here we use structure-informed design to obtain inhibitors of S. aureus IMPDH (SaIMPDH) that have potent antibacterial activity (minimal inhibitory concentrations less than 2 μM) and low cytotoxicity in mammalian cells. The physicochemical properties of the most active compounds were within typical Lipinski/Veber space, suggesting that polarity is not a general requirement for achieving antibacterial activity. Five compounds failed to display activity in mouse models of septicemia and abscess infection. Inhibitor-resistant S. aureus strains readily emerged in vitro. Resistance resulted from substitutions in the cofactor/inhibitor binding site of SaIMPDH, confirming on-target antibacterial activity. These mutations decreased the binding of all inhibitors tested, but also decreased catalytic activity. Nonetheless, the resistant strains had comparable virulence to wild-type bacteria. Surprisingly, strains expressing catalytically inactive SaIMPDH displayed only a mild virulence defect. Collectively these observations question the vulnerability of the enzymatic activity of SaIMPDH as a target for the treatment of S. aureus infections, suggesting other functions of this protein may be responsible for its role in infection.

The microtubule-associated protein tau promotes the stabilization of the axonal cytoskeleton in neurons. In several neurodegenerative diseases, such as Alzheimer's disease, tau has been found to dissociate from microtubules, leading to the formation of pathological aggregates that display an amyloid fibril-like structure. Recent structural studies have shown that the tau filaments isolated from different neurodegenerative disorders have structurally distinct fibril cores that are specific to the disease. These "strains" of tau fibrils appear to propagate between neurons in a prion-like fashion that maintains their initial template structure. In addition, the strains isolated from diseased tissue appear to have structures that are different from those made by the most commonly used in vitro modeling inducer molecule, heparin. The structural differences among strains in different diseases and in vitro-induced tau fibrils may contribute to recent failures in clinical trials of compounds designed to target tau pathology. This study identifies an isoquinoline compound (ANTC-15) isolated from the fungus Aspergillus nidulans that can both inhibit filaments induced by arachidonic acid (ARA) and disassemble preformed ARA fibrils. When compared to a tau aggregation inhibitor currently in clinical trials (LMTX, LMTM, or TRx0237), ANTC-15 and LMTX were found to have opposing inducer-specific activities against ARA and heparin in vitro-induced tau filaments. These findings may help explain the disappointing results in translating potent preclinical inhibitor candidates to successful clinical treatments.

New drugs and new targets are urgently needed to treat tuberculosis. We discovered the D-phenylalanine-benzoxazole Q112 displays potent antibacterial activity against Mycobacterium tuberculosis (Mtb) in multiple media and in macrophage infections. Metabolomic profiling indicates that Q112 has a unique mechanism of action. Q112 perturbs the essential pantothenate/CoA biosynthetic pathway, depleting pantoate while increasing ketopantoate, as would be expected if ketopantoate reductase (KPR) were inhibited. We searched for alternative KPRs since the enzyme annotated as PanE KPR is not essential in Mtb. The ketol-acid reductoisomerase IlvC catalyzes the KPR reaction in the close Mtb relative Corynebacterium glutamicum, but Mtb IlvC does not display KPR activity. We identified the essential protein Rv3603c as an ortholog of PanG KPR, and demonstrated that purified recombinant Rv3603c has KPR activity. Q112 inhibits Rv3603c, explaining the metabolomic changes. Surprisingly, pantothenate does not rescue Q112-treated bacteria, indicating that Q112 has an additional target(s). Q112-resistant strains contain loss-of-function mutations in the twin arginine translocaseTatABC, further underscoring Q112’s unique mechanism of action. Loss of TatABC causes a severe fitness deficit attributed to changes in nutrient uptake, suggesting that Q112 resistance may derive from a decrease in uptake.

This is the accepted manuscript version of the work published in its final form as Pepi, M. J., Chacko, S., Marqus, G. M., Singh, V., Wang, Z., Planck, K., Cullinane, R. T., Meka, P. N., Gollapalli, D. R., Ioerger, T. R., Rhee, K. Y., Cuny, G. D., Boshoff, H. I., & Hedstrom, L. (2022). A d-Phenylalanine-Benzoxazole Derivative Reveals the Role of the Essential Enzyme Rv3603c in the Pantothenate Biosynthetic Pathway of Mycobacterium tuberculosis.ACS Infectious Diseases,8(2), 330-342. https://doi.org/10.1021/acsinfecdis.1c00461 Deposited byshareyourpaper.organdopenaccessbutton.org. We've taken reasonable steps to ensure this content doesn't violate copyright. However, if you think it does you can request a takedown by emailinghelp@openaccessbutton.org.

Many bacterial pathogens, including Staphylococcus aureus, require inosine 5’-monophosphate dehydrogenase (IMPDH) for infection, making this enzyme a promising new target for antibiotics. Although potent selective inhibitors of bacterial IMPDHs have been reported, relatively few have displayed antibacterial activity. Here we use structure-informed design to obtain inhibitors of S. aureus IMPDH (SaIMPDH) that have potent antibacterial activity (minimal inhibitory concentrations less than 2 μM) and low cytotoxicity in mammalian cells. The physicochemical properties of the most active compounds were within typical Lipinski/Veber space, suggesting that polarity is not a general requirement for achieving antibacterial activity. Five compounds failed to display activity in mouse models of septicemia and abscess infection. Inhibitor-resistant S. aureus strains readily emerged in vitro. Resistance resulted from substitutions in the cofactor/inhibitor binding site of SaIMPDH, confirming on-target antibacterial activity. These mutations decreased the binding of all inhibitors tested, but also decreased catalytic activity. Nonetheless, the resistant strains had comparable virulence to wild-type bacteria. Surprisingly, strains expressing catalytically inactive SaIMPDH displayed only a mild virulence defect. Collectively these observations question the vulnerability of the enzymatic activity of SaIMPDH as a target for the treatment of S. aureus infections, suggesting other functions of this protein may be responsible for its role in infection.

New drugs and molecular targets are urgently needed to address the emergence and spread of drug-resistant tuberculosis. Mycobacterium tuberculosis ( Mtb) inosine 5'-monophosphate dehydrogenase 2 ( MtbIMPDH2) is a promising yet controversial potential target. The inhibition of MtbIMPDH2 blocks the biosynthesis of guanine nucleotides, but high concentrations of guanine can potentially rescue the bacteria. Herein we describe an expansion of the structure-activity relationship (SAR) for the benzoxazole series of MtbIMPDH2 inhibitors and demonstrate that minimum inhibitory concentrations (MIC) of ≤1 μM can be achieved. The antibacterial activity of the most promising compound, 17b (Q151), is derived from the inhibition of MtbIMPDH2 as demonstrated by conditional knockdown and resistant strains. Importantly, guanine does not change the MIC of 17b, alleviating the concern that guanine salvage can protect Mtb in vivo. These findings suggest that MtbIMPDH2 is a vulnerable target for tuberculosis.

Herpes simplex virus 1 (HSV-1) has infected more than 80% of the population. Reactivation of the virus causes diseases ranging in severity from benign cold sores to fatal encephalitis. Current treatments involve viral DNA replication inhibitors, but the emergence of drug-resistant mutants is observed frequently, highlighting the need for novel antiviral therapies. Infected cell protein 0 (ICP0) of HSV-1 is encoded by an immediate early gene and plays a fundamental role during infection, because it enables viral gene expression and blocks antiviral responses. One mechanism by which ICP0 functions is through an E3 ubiquitin ligase activity that induces the degradation of targeted proteins. A ΔICP0 virus or mutants with deficiencies in E3 ligase activity cannot counteract beta interferon (IFN-β)-induced restriction of viral infection, are highly immunogenic, are avirulent, and fail to spread. Thus, small molecules interfering with essential and conserved ICP0 functions are expected to compromise HSV-1 infection. We have developed a high-throughput screening assay, based on the autoubiquitination properties of ICP0, to identify small-molecule inhibitors of ICP0 E3 ubiquitin ligase activity. Through a pilot screening procedure, we identified nine compounds that displayed dose-dependent inhibitory effects on ICP0 but not on Mdm2, a control E3 ubiquitin ligase. Following validation, one compound displayed ICP0-dependent inhibition of HSV-1 infection. This compound appeared to bind ICP0 in a cellular thermal shift assay, it blocked ICP0 self-elimination, and it blocked wild-type but not ICP0-null virus gene expression. This scaffold displays specificity and could be used to develop optimized ICP0 E3 ligase inhibitors.IMPORTANCE Since acyclovir and its derivatives were launched for herpesviruses control almost four decades ago, the search for novel antivirals has waned. However, as human life expectancy has increased, so has the number of immunocompromised individuals who receive prolonged treatment for HSV recurrences. This has led to an increase in unresponsive patients due to acquired viral drug resistance. Thus, novel treatments need to be explored. Here we explored the HSV-1 ICP0 E3 ligase as a potential antiviral target because (i) ICP0 is expressed before virus replication, (ii) it is essential for infection in vivo, (iii) it is required for efficient reactivation of the virus from latency, (iv) inhibition of its E3 ligase activity would sustain host immune responses, and (v) it is shared by other herpesviruses. We report a compound that inhibits HSV-1 infection in an ICP0-dependent manner by inhibiting ICP0 E3 ligase activity.

Herpes simplex virus 1 (HSV-1) has infected more than 80% of the population. Reactivation of the virus causes diseases ranging in severity from benign cold sores to fatal encephalitis. Current treatments involve viral DNA replication inhibitors, but the emergence of drug-resistant mutants is observed frequently, highlighting the need for novel antiviral therapies. Infected cell protein 0 (ICP0) of HSV-1 is encoded by an immediate early gene and plays a fundamental role during infection, because it enables viral gene expression and blocks antiviral responses. One mechanism by which ICP0 functions is through an E3 ubiquitin ligase activity that induces the degradation of targeted proteins. A ΔICP0 virus or mutants with deficiencies in E3 ligase activity cannot counteract beta interferon (IFN-β)-induced restriction of viral infection, are highly immunogenic, are avirulent, and fail to spread. Thus, small molecules interfering with essential and conserved ICP0 functions are expected to compromise HSV-1 infection. We have developed a high-throughput screening assay, based on the autoubiquitination properties of ICP0, to identify small-molecule inhibitors of ICP0 E3 ubiquitin ligase activity. Through a pilot screening procedure, we identified nine compounds that displayed dose-dependent inhibitory effects on ICP0 but not on Mdm2, a control E3 ubiquitin ligase. Following validation, one compound displayed ICP0-dependent inhibition of HSV-1 infection. This compound appeared to bind ICP0 in a cellular thermal shift assay, it blocked ICP0 self-elimination, and it blocked wild-type but not ICP0-null virus gene expression. This scaffold displays specificity and could be used to develop optimized ICP0 E3 ligase inhibitors. IMPORTANCE Since acyclovir and its derivatives were launched for herpesviruses control almost four decades ago, the search for novel antivirals has waned. However, as human life expectancy has increased, so has the number of immunocompromised individuals who receive prolonged treatment for HSV recurrences. This has led to an increase in unresponsive patients due to acquired viral drug resistance. Thus, novel treatments need to be explored. Here we explored the HSV-1 ICP0 E3 ligase as a potential antiviral target because (i) ICP0 is expressed before virus replication, (ii) it is essential for infection in vivo, (iii) it is required for efficient reactivation of the virus from latency, (iv) inhibition of its E3 ligase activity would sustain host immune responses, and (v) it is shared by other herpesviruses. We report a compound that inhibits HSV-1 infection in an ICP0-dependent manner by inhibiting ICP0 E3 ligase activity.

Ferrocenyl-Alkyl-Protected Sugar (Fc-Sug) and Ferrocenyl-Oxo-Alkyl-Protected Sugar (Fc-Oxo-Sug) were deposited on the basal plane of Highly Oriented Pyrolytic Graphite (HOPG) using a drop-casting method. Ultrathin films of these molecules were investigated using Atomic Force Microscopy to understand the growth at low coverage. Both molecules are forming highly ordered one-dimensional molecular islands, which are growing from a dimer building block. The dimer and interdimer interactions (along the length of islands) are stabilized by −C2O···H–C hydrogen bonding. Unlike for Fc-Sug, the islands of Fc-Oxo-Sug are extended to tens of micrometers, and the growth is only limited by terrace edges or other islands on the surface. This exceptional growth of islands is understood in terms of an additional −C═O···H–C– hydrogen bonding leading to stronger interdimer interactions along the length of the islands compared to Fc-Sug.

Incorporation of ferrocenyl group to peptides is an efficient method to alter their hydrophobicity. Ferrocenyl group can also act as an electrochemical probe when incorporated onto functional peptides. Most often, ferrocene is incorporated onto peptides post-synthesis via amide, ester or triazole linkages. Stable amino acids containing ferrocene as a C-linked side chain are potentially useful building units for the synthesis of ferrocene-containing peptides. We report here an efficient route to synthesize ferrocene-containing amino acids that are stable and can be used in peptide synthesis. Coupling of 2-ferrocenyl-1,3-dithiane and iodides derived from aspartic acid or glutamic acid using n-butyllithium leads to the incorporation of a ferrocenyl unit to the δ-position or e-position of an α-amino acid. The reduction or hydrolysis of the dithiane group yields an alkyl or an oxo derivative. The usability of the synthesized amino acids is demonstrated by incorporating one of the amino acids in both C-terminus and N-terminus of tripeptides in solution phase.

Diastereoselective reduction of N-protected β-amino ketones does not proceed effectively under the conditions used for chelation controlled reductions of N-alkyl β-amino ketones. A thorough analysis of various conditions required for the stereoselective reduction of γ-aryl-γ-oxo-β-amino alcohols is reported. The products of the syn-selective reduction are used for the preparation of a ceramide trafficking inhibitor HPA-12 and analogues.

An efficient synthesis of deoxygalactonojirimycin and deoxyaltronojirimycin through the use of proline catalyzed asymmetric α-aminoxylation of a higher homologue of Garner's aldehyde, derived from l-aspartic acid, is reported. The method is also used for a highly diastereoselective synthesis of the N-Boc derivative of (2S,3S)-3-hydroxypipecolic acid. The configuration of the proline catalyst used for the asymmetric aminoxylation step ultimately controls the absolute configuration of three adjacent stereogenic centers in the final products.

A very effective one-pot three component reaction of 4-bromobutanal or 5-bromopentanal, acetone, and p -anisidine catalyzed by proline is reported. A three component Mannich reaction followed by cyclization leading to the synthesis of 2-substituted pyrrolidine and piperidine derivatives through simultaneous formation of two C–N and one C–C bond is reported. The usefulness of the reaction is demonstrated by the synthesis of (±)coniine, (±)pelletrine, (±)sedridine, and (±)allosedridine.

An efficient method for covalently linking of cellulose and clay using a click chemistry based strategy is reported. Azide and alkynyl derivatives of silane were synthesized and used for silanization of cellulose and clay respectively. Functionalized cellulose and clay were then coupled using Cu(I) catalyzed azide–alkyne cycloaddition reaction, resulting in a covalent linkage between them. Successful synthesis of the silane derivates was established using Fourier transform infrared (FTIR) and nuclear magnetic resonance. Silanization of cellulose and clay with azide and alkynyl derivatives and the formation of a triazole linkage were confirmed using FTIR.

Francisella tularensis is the causative agent of tularemia and a potential biowarfare agent. The virulence of F. tularensis is decreased by deletion of guaB , the gene encoding IMP dehydrogenase (IMPDH), suggesting that this enzyme is a target for antibacterial design. Here we report that F. tularensis growth is blocked by inhibitors of bacterial IMPDHs. Seventeen compounds from two different frameworks, designated the D and Q series, display antibacterial activities with MICs of guaB . These observations suggest that antibacterial activity derives from inhibition of another target(s). Moreover, D compounds display antibacterial activity only against F. tularensis , suggesting the presence of a unique target or uptake mechanism. A Δ guaB mutant resistant to compound D73 contained a missense mutation (Gly45Cys) in nuoB , which encodes a subunit of bacterial complex I. Overexpression of the nuoB mutant conferred resistance to D73 in both wild-type and Δ guaB strains. This strain was not resistant to Q compounds, suggesting that a different off-target mechanism operates for these compounds. Several Q compounds are also effective against Mycobacterium tuberculosis , in which a second target has also been implicated, in addition to IMPDH. The fortuitous presence of multiple targets with overlapping structure-activity relationships presents an intriguing opportunity for the development of robust antibiotics that may avoid the emergence of resistance.

The purine nucleotide pathways are tempting targets for antibiotic design. However, the vulnerability of these potential targets is questioned by the potential redundancy of the de novo biosynthesis and salvage pathways questions. Indeed, genetic experiments suggest that the vulnerability of these enzymes varies with pathogen and environmental niche. More target validation in the form of conditional knockouts could provide the confidence needed to launch drug discovery efforts. The purine conversion pathways are required for both de novo synthesis and salvage, suggesting that these four enzymes, adenylosuccinate synthetase, adenylosuccinate lyase, IMP dehydrogenase and GMP synthetase, are the better candidates. Even here the similarity of the active sites in pathogen and host enzymes can be discouraging. To date, only inhibitors of IMP dehydrogenase have been pursued with a significant effort. Mammalian and bacterial IMP dehydrogenases have dramatically different cofactor binding sites that can be exploited to identify selective inhibitors. Antibacterial activity has been reported against Gram positive pathogens and Mycobacterium tuberculosis, but no compound has achieved the critical milestone of activity in an animal model of infection.

An enantiospecific route to the synthesis of tetrahydroquinoline alkaloids (–)-cuspareine and (–)-galipinine is reported. Coupling of an iodide derivative of D-serine with aromatic dithianes and Pd-catalyzed intramolecular C–N coupling are the key steps in the synthesis.

Treatment of the 1,3-dithiane derivative of the Garner aldehyde with alkyl halides in the presence of n -butyllithium resulted in a ring-opening reaction to give differentially substituted propane-1,3-dithioethers, instead of the expected 2,2-dialkylated 1,3-dithiane products. The corresponding 1,3-dithiolane derivative also reacted in a similar manner. Such ring-opening reactions of dithiane or dithiolane derivatives are rare. A detailed study of this unusual reaction was carried out and a possible mechanism is proposed.

γ-Oxo α-amino acids and γ-aryl α-amino acids are compounds with very interesting biological properties and are active components of many drug molecules. Oxo amino acids are also used as synthetic precursors for a number of unnatural amino acids and amino alcohols. We report a very efficient synthesis of such compounds through the coupling of aromatic dithianes, prepared from aromatic aldehydes and an iodide derivative of serine. The dithiane groups in compounds thus obtained can be either hydrolyzed or reduced to generate 4-oxo-4-aryl or 4-aryl 2-amino butanol derivatives, respectively, which, on further transformations, can be converted into the title compounds. Starting with L-serine provides the corresponding D-amino acids with complete enantiopurity. The reported method is economically viable and complements the existing methods, which rely largely on cross-coupling reactions.

Diastereoselective reduction of N-protected β-amino ketones does not proceed effectively under the conditions used for chelation controlled reductions of N-alkyl β-amino ketones. A thorough analysis of various conditions required for the stereoselective reduction of γ-aryl-γ-oxo-β-amino alcohols is reported. The products of the syn-selective reduction are used for the preparation of a ceramide trafficking inhibitor HPA-12 and analogues.

Incorporation of ferrocenyl group to peptides is an efficient method to alter their hydrophobicity. Ferrocenyl group can also act as an electrochemical probe when incorporated onto functional peptides. Most often, ferrocene is incorporated onto peptides post-synthesis via amide, ester or triazole linkages. Stable amino acids containing ferrocene as a C-linked side chain are potentially useful building units for the synthesis of ferrocene-containing peptides. We report here an efficient route to synthesize ferrocene-containing amino acids that are stable and can be used in peptide synthesis. Coupling of 2-ferrocenyl-1,3-dithiane and iodides derived from aspartic acid or glutamic acid using n-butyllithium leads to the incorporation of a ferrocenyl unit to the δ-position or ε-position of an α-amino acid. The reduction or hydrolysis of the dithiane group yields an alkyl or an oxo derivative. The usability of the synthesized amino acids is demonstrated by incorporating one of the amino acids in both C-terminus and N-terminus of tripeptides in solution phase.

Piperidine derivative (IVb) serves as the substrate for the preparation of racemic N-Boc-coniine (Va), racemic pelletrine (Vb) and racemic sedridine or allosedridine (Vc), respectively.

γ-Oxo α-amino acids and γ-aryl α-amino acids are compounds with very interesting biological properties and are active components of many drug molecules. Oxo amino acids are also used as synthetic precursors for a number of unnatural amino acids and amino alcohols. We report a very efficient synthesis of such compounds through the coupling of aromatic dithianes, prepared from aromatic aldehydes and an iodide derivative of serine. The dithiane groups in compounds thus obtained can be either hydrolyzed or reduced to generate 4-oxo-4-aryl or 4-aryl 2-amino butanol derivatives, respectively, which, on further transformations, can be converted into the title compounds. Starting with L-serine provides the corresponding D-amino acids with complete enantiopurity. The reported method is economically viable and complements the existing methods, which rely largely on cross-coupling reactions.