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Abstract Adaptive laboratory evolution experiments provide a controlled context in which the dynamics of selection and adaptation can be followed in real-time at the single-nucleotide level. And yet this precision introduces hundreds of degrees-of-freedom as genetic changes accrue in parallel lineages over generations. On short timescales, physiological constraints have been leveraged to provide a coarse-grained view of bacterial gene expression characterized by a small set of phenomenological parameters. Here, we ask whether this same framework, operating at a level between genotype and fitness, informs physiological changes that occur on evolutionary timescales. Using a strain adapted to growth in glucose minimal medium, we find that the proteome is substantially remodeled over 40 000 generations. The most striking change is an apparent increase in enzyme efficiency, particularly in the enzymes of lower-glycolysis. We propose that deletion of metabolic flux-sensing regulation early in the adaptation results in increased enzyme saturation and can account for the observed proteome remodeling.

In the context of our ongoing studies on chromane derivatives as inhibitors of the salicylate synthase from M. tuberculosis, we isolated a new, unexpected compound from the cyclization of 3-(propargyloxy)-5-benzyloxy-benzoic acid methyl ester. Its molecular structure was elucidated by means of 1D and 2D NMR analyses, FT-IR, ESI-MS, and HRMS.

Abstract Quantifying the contribution of individual molecular components to complex cellular processes is a grand challenge in systems biology. Here we establish a general theoretical framework (Functional Decomposition of Metabolism, FDM) to quantify the contribution of every metabolic reaction to metabolic functions, e.g. the synthesis of biomass building blocks. FDM allowed for a detailed quantification of the energy and biosynthesis budget for growing Escherichia coli cells. Surprisingly, the ATP generated during the biosynthesis of building blocks from glucose almost balances the demand from protein synthesis, the largest energy expenditure known for growing cells. This leaves the bulk of the energy generated by fermentation and respiration unaccounted for, thus challenging the common notion that energy is a key growth-limiting resource. Moreover, FDM together with proteomics enables the quantification of enzymes contributing towards each metabolic function, allowing for a first-principle formulation of a coarse-grained model of global protein allocation based on the structure of the metabolic network.

MbtI from Mycobacterium tuberculosis (Mtb) is a Mg2+-dependent salicylate synthase, belonging to the chorismate-utilizing enzyme (CUE) family. As a fundamental player in iron acquisition, MbtI promotes the survival and pathogenicity of Mtb in the infected host. Hence, it has emerged in the last decade as an innovative, potential target for the anti-virulence therapy of tuberculosis. In this context, 5-phenylfuran-2-carboxylic acids have been identified as potent MbtI inhibitors. The first co-crystal structure of MbtI in complex with a member of this class was described in 2020, showing the enzyme adopting an open configuration. Due to the high mobility of the loop adjacent to the binding pocket, large portions of the amino acid chain were not defined in the electron density map, hindering computational efforts aimed at structure-driven ligand optimization. Herein, we report a new, high-resolution co-crystal structure of MbtI with a furan-based derivative, in which the closed configuration of the enzyme allowed tracing the entirety of the active site pocket in the presence of the bound inhibitor. Moreover, we describe a new crystal structure of MbtI in open conformation and in complex with the known inhibitor methyl-AMT, suggesting that in vitro potency is not related to the observed enzyme conformation. These findings will prove fundamental to enhance the potency of this series via rational structure-based drug-design approaches.

The ever-evolving research in the field of antitubercular agents has led to the identification of several new potential drug classes. Among them, 5-phenyl-furan-2-carboxylic acids have emerged as innovative potential therapeutics, targeting iron acquisition in mycobacterial species. In our efforts to characterize the molecular interactions between these compounds and their protein target (MbtI from M. tuberculosis) by means of co-crystallization experiments, we unexpectedly obtained the structure of 5-(4-nitrophenyl)furan-2-carboxylic acid (1). Herein, we describe the preparation of the compound and its analysis by 1H NMR, 13C NMR, HRMS, and SC-XRD.

5-Phenyl-furan-2-carboxylic acids have emerged as a new, promising class of antimycobacterial agents that have the ability to interfere with iron homeostasis. Considering the lack of structural data on these compounds, we analyzed the crystal of a fluorinated ester derivative of 5-(4-nitrophenyl)furan-2-carboxylic acid, one of the most potent candidates in the series. Here, we describe the preparation of methyl 5-(2-fluoro-4-nitrophenyl)furan-2-carboxylate (1) and its analysis by 1H-NMR, 13C-NMR, HRMS, and SC-XRD.

Biofilm-dwelling cells endure adverse conditions, including oxidative imbalances. The NADH:quinone oxidoreductase enzyme WrbA has a crucial role in the mechanism of action of antibiofilm molecules such as ellagic and salicylic acids. This study aimed to exploit the potential of the WrbA scaffold as a valuable target for identifying antibiofilm compounds at non-lethal concentrations. A three-dimensional computational model, based on the published WrbA structure, was used to screen natural compounds from a virtual library of 800,000 compounds. Fisetin, morin, purpurogallin, NZ028, and NZ034, along with the reference compound ellagic acid, were selected. The antibiofilm effect of the molecules was tested at non-lethal concentrations evaluating the cell-adhesion of wild-type and WrbA-deprived Escherichia coli strains through fluorochrome-based microplate assays. It was shown that, except for NZ028, all of the selected molecules exhibited notable antibiofilm effects. Purpurogallin and NZ034 showed excellent antibiofilm performances at the lowest concentration of 0.5 μM, in line with ellagic acid. The observed loss of activity and the level of reactive oxygen species in the mutant strain, along with the correlation with terms contributing to the ligand-binding free energy on WrbA, strongly indicates the WrbA-dependency of purpurogallin and NZ034. Overall, the molecular target WrbA was successfully employed to identify active compounds at non-lethal concentrations, thus revealing, for the first time, the antibiofilm efficacy of purpurogallin and NZ034.

Bispidines are a family of ligands that plays a pivotal role in various areas of coordination chemistry, with applications in medicinal chemistry, molecular catalysis, coordination polymers synthesis, and molecular magnetism. In the present work, triazole moieties were introduced using the CuAAC click-reaction, with the aim of expanding the number of coordination sites on the bispidine core. The 1,2,3-triazole rings were thus synthesized on propargyl-derived bispidines after reaction with different alkyl azides. The new class of triazole-bispidines was characterized, and their chelation capabilities were evaluated with different metals through NMR titration, ESI-MS spectrometry, and single-crystal X-ray diffraction (SC-XRD). Finally, the suitability of these molecules as metal ligands for the catalytic Henry reaction was demonstrated with copper and zinc.

Bacterial biofilm is a major contributor to the persistence of infection and the limited efficacy of antibiotics. Antibiofilm molecules that interfere with the biofilm lifestyle offer a valuable tool in fighting bacterial pathogens. Ellagic acid (EA) is a natural polyphenol that has shown attractive antibiofilm properties. However, its precise antibiofilm mode of action remains unknown. Experimental evidence links the NADH:quinone oxidoreductase enzyme WrbA to biofilm formation, stress response, and pathogen virulence. Moreover, WrbA has demonstrated interactions with antibiofilm molecules, suggesting its role in redox and biofilm modulation. This work aims to provide mechanistic insights into the antibiofilm mode of action of EA utilizing computational studies, biophysical measurements, enzyme inhibition studies on WrbA, and biofilm and reactive oxygen species assays exploiting a WrbA-deprived mutant strain of Escherichia coli. Our research efforts led us to propose that the antibiofilm mode of action of EA stems from its ability to perturb the bacterial redox homeostasis driven by WrbA. These findings shed new light on the antibiofilm properties of EA and could lead to the development of more effective treatments for biofilm-related infections.

PPARγ represents a key target for the treatment of type 2 diabetes and metabolic syndrome. To avoid serious adverse effects related to the PPARγ agonism profile of traditional antidiabetic drugs, a new opportunity is represented by the development of molecules acting as inhibitors of PPARγ phosphorylation by the cyclin-dependent kinase 5 (CDK5). Their mechanism of action is mediated by the stabilization of the PPARγ β-sheet containing Ser273 (Ser245 in PPARγ isoform 1 nomenclature). In this paper, we report the identification of new γ-hydroxy-lactone-based PPARγ binders from the screening of an in-house library. These compounds exhibit a non-agonist profile towards PPARγ, and one of them prevents Ser245 PPARγ phosphorylation by acting mainly on PPARγ stabilization and exerting a weak CDK5 inhibitory effect.

Targeting pathogenic mechanisms, rather than essential processes, represents a very attractive approach for the development of new antimycobacterial drugs. In this context, iron acquisition routes have recently emerged as potentially druggable pathways. However, the importance of siderophore biosynthesis in the virulence and pathogenicity of M. abscessus (Mab) is still poorly understood. In this study, we investigated the Salicylate Synthase (SaS) of Mab as an innovative molecular target for the development of inhibitors of siderophore production. Notably, Mab-SaS does not have any counterpart in human cells, making it an interesting candidate for drug discovery. Starting from the analysis of the binding of a series of furan-based derivatives, previously identified by our group as inhibitors of MbtI from M. tuberculosis (Mtb), we successfully selected the lead compound 1, exhibiting a strong activity against Mab-SaS (IC50 ≈ 5 µM). Computational studies characterized the key interactions between 1 and the enzyme, highlighting the important roles of Y387, G421, and K207, the latter being one of the residues involved in the first step of the catalytic reaction. These results support the hypothesis that 5-phenylfuran-2-carboxylic acids are also a promising class of Mab-SaS inhibitors, paving the way for the optimization and rational design of more potent derivatives.

Herein, we report the preparation of methyl 6-methyl-4-(4-nitrophenyl)-2-oxo-1,2-dihydropyrimidine-5-carboxylate 2, obtained by the regioselective oxidative dehydrogenation of the dihydropyrimidine derivative 1 in the presence of cerium ammonium nitrate. The structure of compound 2 was investigated by single-crystal X-ray diffraction (SC-XRD), which allowed the determination of its tautomeric form. Moreover, the presence of non-covalent interactions and their impact on the crystal structure were analyzed. To better characterize the intermolecular contacts, the Hirshfeld surface and enrichment ratio analyses were performed. Furthermore, the antimycotic activity of compounds 1 and 2 was investigated against Candida albicans, Aspergillus flavus, and Aspergillus niger, and their efficacy was compared to that of fluconazole. Computational investigations on the putative target of the compounds provided insights to explain the better activity of 2 with respect to its synthetic precursor.

Tuberculosis (TB) causes millions of deaths every year, ranking as one of the most dangerous infectious diseases worldwide. Because several pathogenic strains of M. tuberculosis (Mtb) have developed resistance against most of the established anti-TB drugs, new therapeutic options are urgently needed. An attractive target for the development of new anti-TB agents is the salicylate synthase MbtI, the first enzyme of the mycobacterial siderophore biochemical machinery, absent in human cells. In this work, a set of analogues of 5-(3-cyanophenyl)furan-2-carboxylic acid (I), the most potent MbtI inhibitor identified to date, was synthesized, characterized, and tested to further elucidate the structural requirements for achieving an efficient MbtI inhibition and potent antitubercular activity. The structure–activity relationships (SAR) discussed herein evidenced the importance of the side chain linked to the phenyl moiety to improve the in vitro antimycobacterial activity. In detail, 1f emerged as the most effective analogue against the pathogen, acting without cytotoxicity issues. To deepen the understanding of its mechanism of action, we established a fluorescence-based screening test to quantify the pathogen infectivity within host cells, using MPI-2 murine cells, a robust surrogate for alveolar macrophages. The set-up of the new assay demonstrates significant potential to accelerate the discovery of new anti-TB drugs.

SIRT5 is a member of the Sirtuin family, a class of deacetylating enzymes consisting of seven isoforms, involved in the regulation of several processes, including gene expression, metabolism, stress response, and aging. Considering that the anomalous activity of SIRT5 is linked to many pathological conditions, we present herein an overview of the most interesting modulators, with the aim of contributing to further development in this field.

Abstract We initiate a systematic study of type II orientifold flux vacua in three dimensions including gauge and metric fluxes, O-planes and D-branes. We derive simple flux models (we dub them RSTU-models) that admit a description in terms of gauged supergravities with half-maximal N $$ \mathcal{N} $$ = 8 supersymmetry in three dimensions. As a landscape appetizer, we present various multi-parametric families of supersymmetric and non-supersymmetric AdS3 and Mkw3 vacua. Remarkably, negative masses turn out to be always absent in the spectrum of scalar fluctuations, thus making all the vacua perturbatively stable within half-maximal supergravity. We provide examples of non-supersymmetric type IIB AdS3 flux vacua which feature parametrically-controlled scale separation and come along with integer-valued conformal dimensions of the would-be dual CFT2 operators. We also comment on the implications of our results in light of the Swampland Program.

Abstract We study supersymmetric AdS $$_3$$ 3 flux vacua of massive type-IIA supergravity on anisotropic G2 orientifolds. Depending on the value of the $$F_4$$ F 4 flux the seven-dimensional compact space can either have six small and one large dimension such that the “external” space is scale-separated and effectively four-dimensional, or all seven compact dimensions small and parametrically scale-separated from the three external ones. Within this setup we also discuss the Distance Conjecture (including appropriate D4-branes), and highlight that such vacua provide a non-trivial example of the so-called strong Spin-2 Conjecture.

Abstract We discuss minimally supersymmetric AdS3 flux vacua of massive type IIA supergravity on G2-orientifolds. We find that configurations with broken scale-separation can be within finite distance from scale-separated ones, while both remain at large volume, weak coupling and have moduli stabilization. The transition is achieved with the use of a D4-brane modulus, which allows the F 4 flux to jump, and has an effective potential always accessible to the three-dimensional low-energy theory. Our analysis further allows us to check the distance conjecture quantitatively, as we can track explicitly the masses of the KK modes.

Abstract We analyze the standard fermionic 4D Volkov–Akulov action with N global non-linear supersymmetries. We find that the stationary points of the system are described by an effective potential, written in terms of two composite real scalar fields, which approaches the exact quantum effective potential in the large N limit. We identify the existence of at least two stationary points, one representing the original supersymmetry breaking configuration and the other one corresponding to goldstino condensation, where supersymmetry is restored in the deep IR.

Abstract We investigate the possible appearance of composite states of the goldstino in models with four-dimensional non-linear supersymmetry and we provide a description of their dynamics in terms of a Kähler potential and a superpotential. Our analysis shows that the critical point corresponding to the Volkov-Akulov model is unstable. Similarly, we find that the uplifted stable de Sitter critical point of the KKLT model is shifted and acquires a tachyonic instability. Our findings indicate the existence of a potentially dangerous instability shared by all anti-brane uplifts.

Easy-to-use evaluation of Range Of Motion (ROM) during walking is necessary to make decisions during neurological rehabilitation programs and during follow-up visits in clinical and remote settings. This study discussed goniometer applications (DrGoniometer and Angles - Video Goniometer) that measure knee joint ROM during walking through smartphone cameras. The primary aim of the study is to test the inter-rater and intra-rater reliability of the collected measurements as well as their concurrent validity with an electro-goniometer. The secondary aim is to evaluate the usability of the two mobile applications. A total of 22 patients with Parkinson’s disease (18 males, age 72 (8) years), 22 post-stroke patients (17 males, age 61 (13) years), and as many healthy volunteers (8 males, age 45 (5) years) underwent knee joint ROM evaluations during walking. Clinicians and inexperienced examiners used the two mobile applications to calculate the ROM, and then rated their perceived usability through the System Usability Scale (SUS). Intraclass correlation coefficients (ICC) and correlation coefficients (corr) were calculated. Both applications showed good reliability (ICC > 0.69) and validity (corr > 0.61), and acceptable usability (SUS > 68). Smartphone-based video goniometers could be used to assess the knee ROM during walking in neurological patients, because of their acceptable degree of reliability, validity and usability.