Norbert Reiling

The proteasome activator PA200 binds to the catalytic core of both standard proteasome (s20S) and the immunoproteasome (i20S); however, whether PA200 uses the same mechanisms to activate i20S remains unknown. In this work, the cryo-EM structures of singly- and doubly-capped i20S–PA200 complexes, combined with complementary in vitro biochemical assays, show that binding of the first PA200 induces allosteric bending of the i20S and widens the opposite α-ring, promoting higher PA200 occupancy and stronger activation compared to the s20S. PA200 also selectively modulates i20S proteolytic activity by enhancing peptide production and shifting cleavage specificity toward caspase-like activity. In cells and tissues co-expressing PA200, s20S, and i20S, PA200 preferentially associates with the i20S. Moreover, PA200 and i20S catalytic subunits are differentially regulated, with PA200 playing a potential role in regulating the i20S subunits’ expression. Overall, these findings suggest that PA200 contributes to the regulation of i20S function and dynamics.

To tackle the emerging resistance against existing antibiotics, we screened natural-product (NP) libraries against two underexploited target enzymes from the 2C-methyl-d-erythritol 4-phosphate (MEP) pathway, namely, Mycobacterium tuberculosis DXPS and Escherichia coli IspD. We have chosen these two enzymes due to the availability of the crystal structures that helped to elucidate the putative binding modes of the NPs identified. The screening of a NP collection led to the discovery of myxobacteria-derived maracen A and Streptomyces-derived polyketomycin as the first NPs targeting these enzymes.

Simplified amide IspE inhibitors restore potency against Klebsiella pneumoniae and Escherichia coli , offering synthetically accessible antibacterial hits.

Amides of squaric acid are new drug candidates with activity against mycobacteria. Like the approved drug bedaquiline, these compounds achieve efficacy by inhibiting mycobacterial ATP synthase. However, squaramides have a different binding site than bedaquiline and possess the potential to inhibit bedaquiline-resistant strains. We developed an optimized synthesis for monoamino-substituted squaric acid analogues. Guided by an atomic model of a squaramide compound bound to its target, we synthesized 31 new monoamino/diamino-substituted squaric acid derivates. The efficacy of these compounds was determined in whole-cell assays against Mycobacterium tuberculosis and Mycobacterium avium. The molecular target was confirmed with measurement of inhibition of Mycobacterium smegmatis ATP synthase and by using M. tuberculosis strains that modulate the expression of ATP synthase. Compared to earlier squaramides, several analogues demonstrated micromolar activity against M. tuberculosis, improved microsomal stability in vitro, and reduced cytotoxicity. These properties contribute to the preclinical development of this class of compound.

The rise of multidrug-resistant pathogens, such as Staphylococcus aureus and Mycobacterium tuberculosis, underscores an urgent need for therapeutic innovation. The antibiotic development pipeline targeting these bacteria is critically limited, with most discovered candidates exhibiting structurally similar features of prominent chemical entities and with well-established molecular targets or binding modes. The myxobacterial α-pyrone antibiotics, myxopyronins, represent a highly promising compound class due to their ability to inhibit RNA polymerase by binding to the “switch region”, a distinct binding site to that of standard-of-care antibiotics. Mutasynthesis, leveraging engineered microorganisms and tailored precursors, provides a viable alternative to total synthesis for generating novel derivatives. This study utilized a heterologous expression system in Myxococcus xanthus DK1622 to generate analogs. Two carrier protein domain mutants were engineered to facilitate mutasynthesis-based production of structurally diverse derivatives. A trifluoromethyl-modified analog, once accessible only through total synthesis but now obtained via mutasynthesis, exhibits potent antimicrobial activity against Gram-positive pathogens including Mycobacterium tuberculosis and favorable in vitro absorption, distribution, metabolism, excretion and toxicity properties. These findings highlight a promising pathway for developing optimized α-pyrone antibiotics to address the global antimicrobial-resistance crisis.

Tuberculosis, caused by Mycobacterium tuberculosis (Mtb), remains a leading cause of mortality worldwide. Proteins involved in lipid metabolism, such as the monoacylglycerol lipase Rv0183, play critical roles during both the active and dormant phases of Mtb and present novel targets for therapeutic intervention. Through high-throughput screening at the European Lead Factory, we identified a novel chemotype characterized by a hydroxypyrrolidine ring, which demonstrated potent inhibition of Rv0183 and promising results in whole cell bacterial studies. Subsequent co-crystallization studies of this chemotype with Rv0183 revealed non-covalent interactions within the lipase’s binding pocket, elucidating the inhibitory mechanism. Comparative analysis, augmented by AI-driven 3D-point-cloud approaches, distinguished Rv0183’s ligand-binding cavity from that of human monoacylglycerol lipase, implying the possibility for species-selective inhibition. This selectivity was further supported by molecular docking simulations which validated the experimental binding affinities and predicted strong, specific binding modes. Our study presents not only the structural basis for the inhibition of Rv0183 by these novel hydroxypyrrolidine-based inhibitors but also demonstrates the utility of integrating computational and empirical methods to achieve species-specific targeting. This approach could minimize off-target effects in humans, marking a significant step toward developing more effective antitubercular therapies. The potential to selectively inhibit Mtb in its dormant state could lead to treatments that prevent the persistence and resurgence of the disease, addressing a crucial gap in the fight against tuberculosis.

The proteasome activator PA200 binds to the catalytic core of both standard proteasome (s20S) and the immunoproteasome (i20S); however, whether PA200 uses the same mechanisms to activate i20S remains unknown. Our cryo-EM structures of the singly- and doubly-capped i20S-PA200 complexes and in vitro assays revealed that binding of the first PA200 induces an allosteric widening of the opposite unbound α-ring, resulting in a higher binding occupancy of the i20S and its stronger activation compared to the s20S. We also showed that in cells and tissues that express PA200, s20S and i20S, PA200 interacts preferably with i20S. Intriguingly, the expression of PA200 and the catalytic subunits of the i20S are differentially regulated, with PA200 playing a potential role in controlling the i20S subunits’ expression. This suggests that i20S function is fine-tuned by differential expression of PA200, and reveals an additional layer of i20S regulation.

The rise of multidrug-resistant pathogens, such as Staphylococcus aureus and Mycobacterium tuberculosis, underscores an urgent need for therapeutic innovation. The antibiotic development pipeline targeting these bacteria is critically limited, with most discovered candidates exhibiting structurally similar features of prominent chemical entities, with well-established molecular targets or binding modes. The myxobacterial α-pyrone antibiotics, myxopyronins, represent a highly promising compound class due to their ability to inhibit RNA polymerase by binding to the "switch region", a distinct binding site to that of standard-of-care antibiotics. Although total synthesis has enabled access to the natural products, this strategy faces significant limitations to generate analogs. Mutasynthesis, leveraging engineered microorganisms and tailored precursors, provides a viable alternative to generate novel derivatives. This study utilized a heterologous expression system in Myxococcus xanthus DK1622 to generate analogs. Two carrier protein domain mutants were engineered to facilitate mutasynthesis-based production of structurally diverse derivatives. A trifluoromethyl-modified analog demonstrated potent antimicrobial activity against Gram-positive pathogens including Mycobacterium tuberculosis and favorable in vitro absorption, distribution, metabolism, excretion and toxicity properties. These findings highlight a promising pathway for developing optimized α pyrone antibiotics to address the global antimicrobial-resistance crisis.

In this study, we synthesized a library of eNTRy-rule-compliant compounds by introducing ionizable nitrogen atoms to an antimalarial compound. These positively charged derivatives gained activity against both Gram-negative and -positive bacteria, Mycobacterium tuberculosis, and boosted Plasmodium falciparum inhibition to the double-digit nanomolar range. Overcoming and remaining inside the cell envelope of Gram-negative bacteria (GNB) is one of the major difficulties in antibacterial drug discovery and development. The eNTRy rules (N = ionizable nitrogen, T = low three-dimensionality, R = rigidity) can be a useful structural guideline to improve accumulation of small molecules in GNB. With the aim of unlocking Gram-negative activity, we added amines and (cyclic) N-alkyl guanidines to an already flat and rigid pyrazole-amide class as a representative example for our investigation. To test their performance, we compared these eNTRy-rule-compliant compounds to closely related noncompliant ones through phenotypic screening of various pathogens (P. falciparum, Escherichia coli, Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumoniae, and M. tuberculosis), obtaining a handful of broad-spectrum hits. The results support the working hypothesis and even extend its applicability. The studied pyrazole-amide class adheres to the eNTRy rules; noncompliant compounds do not kill any of the bacteria tested, while compliant compounds largely showed growth inhibition of Gram-negative, -positive, and M. tuberculosis bacteria in the single-digit micromolar range.

Mycobacterium tuberculosis is well adapted to survive and persist in the infected host, escaping the host’s immune response. Since polyamines such as spermine, which are synthesized by infected macrophages, are able to inhibit the growth of M. tuberculosis , the pathogen needs strategies to cope with these toxic metabolites. The actinomycete Streptomyces coelicolor , a close relative of M. tuberculosis, makes use of a gamma-glutamylation pathway to functionally neutralize spermine. We therefore considered whether a similar pathway would be functional in M. tuberculosis . In the current study, we demonstrated that M. tuberculosis growth was inhibited by the polyamine spermine. Using in vitro enzymatic assays we determined that GlnA3 _Mt (Rv1878) possesses genuine gamma-glutamylspermine synthetase catalytic activity. We further showed that purified His-Strep-GlnA3 _Mt , as well as native GlnA3 _Mt , prefer spermine as a substrate over putrescine, cadaverine, spermidine, or other monoamines and amino acids, suggesting that GlnA3 _Mt may play a specific role in the detoxification of the polyamine spermine. However, the deletion of the glnA3 gene in M. tuberculosis did not result in growth inhibition or enhanced sensitivity of M. tuberculosis in the presence of high spermine concentrations. Gene expression analysis of spermine-treated M. tuberculosis revealed no difference in the level of glnA3 _Mt expression relative to untreated cells, whereas a gene encoding a previously characterized efflux pump (Mmr; rv3065 ) was significantly upregulated. This suggests that bacterial survival under elevated spermine concentrations can not only be achieved by detoxification of spermine itself but also by mechanisms resulting in decreased spermine levels in the bacteria. IMPORTANCE: Upon Mycobacterium tuberculosis infection macrophages synthesize the polyamine spermine, which at elevated concentrations is toxic for M. tuberculosis . Based on our investigations of spermine resistance in the closely related actinomycete Streptomyces coelicolor , we hypothesized that the glutamylspermine synthetase GlnA3 may be responsible for the resistance of M. tuberculosis against toxic spermine. Here we show that GlnA3 _Mt can indeed covalently modify spermine via glutamylation. However, GlnA3 _Mt is probably not the only resistance mechanism since a glnA3 null mutant of M. tuberculosis can survive under spermine stress. Gene expression studies suggest that an efflux pump may participate in resistance. Thus a combination of GlnA3 _Mt and specific efflux pumps acting as putative spermine transporters may constitute an active spermine-detoxification system in M. tuberculosis .