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Enzymatic pockets such as those of histone deacetylases (HDACs) are among the most favored targets for drug development. However, enzymatic inhibitors often exhibit low selectivity and high toxicity due to targeting multiple enzyme paralogs, which are often involved in distinct multisubunit complexes. Here, we report the discovery and characterization of a non-enzymatic small molecule inhibitor of HDAC transcriptional repression functions with comparable anti-tumor activity to the enzymatic HDAC inhibitor Vorinostat, and anti-psychedelic activity of anHDAC2knockoutin vivo. We highlight that these phenotypes are achieved while modulating the expression of 20- and 80-fold fewer genes than enzymatic and genetic inhibition in the respective models. Thus, by achieving the same biological outcomes as established therapeutics while impacting a dramatically smaller number of genes, inhibitors of protein-protein interactions can offer important advantages in improving the selectivity of epigenetic modulators.GRAPHICAL ABSTRACT

Among the functions exerted by eukaryotic lactate dehydrogenases, it is of importance the generation of lactate in muscles subjected to fatigue or to limited oxygen availability, with both these conditions triggering a decrease of cellular pH. However, the mutual dependence between lactate dehydrogenase (LDH) catalytic action and lactic acidosis is far from being fully understood. Here we show that the tetrameric LDH from rabbit skeletal muscle undergoes allosteric transitions as a function of pH, i.e. the enzyme obeys Michaelis-Menten kinetics at neutral or slightly alkaline pH values, and features sigmoidal kinetics at pH 6.5 or lower. Remarkably, we also report that a significant dissociation of tetrameric rabbit LDH occurs under acidic conditions, with pyruvate/NAD+ or citrate counteracting this effect. Moreover, citrate strongly activates rabbit LDH, inducing the enzyme to feature Michaelis-Menten kinetics. Further, using primary rabbit skeletal muscle cells we tested the generation of lactate as a function of pH, and we detected a parallel decrease of cytosolic pH and secretion of lactate. Overall, our observations indicate that lactic acidosis is antagonized by LDH dissociation, the occurrence of which is regulated by citrate and by allosteric transitions of the enzyme induced by pyruvate.

The aerobic energetic metabolism of eukaryotic cells relies on the glycolytic generation of pyruvate, which is subsequently channelled to the oxidative phosphorylation taking place in mitochondria. However, under conditions limiting oxidative phosphorylation, pyruvate is coupled to alternative energetic pathways, e.g. its reduction to lactate catalyzed by lactate dehydrogenases (LDHs). This biochemical process is known to induce a significant decrease in cytosolic pH, and is accordingly denoted lactic acidosis. Nevertheless, the mutual dependence of LDHs action and lactic acidosis is far from being fully understood. Using human LDH-A, here we show that when exposed to acidic pH this enzyme is subjected to homotropic allosteric transitions triggered by pyruvate. Conversely, human LDH-A features Michaelis–Menten kinetics at pH values equal to 7.0 or higher. Further, citrate, isocitrate, and malate were observed to activate human LDH-A, both at pH 5.0 and 6.5, with citrate and isocitrate being responsible for major effects. Dynamic light scattering (DLS) experiments revealed that the occurrence of allosteric kinetics in human LDH-A is mirrored by a consistent dissociation of the enzyme tetramer, suggesting that pyruvate promotes tetramer association under acidic conditions. Finally, using the human liver cancer cell line HepG2 we isolated cells featuring cytosolic pH equal to 7.3 or 6.5, and we observed a concomitant decrease in cytosolic pH and lactate secretion. Overall, our observations indicate the occurrence of a negative feedback between lactic acidosis and human LDH-A activity, and a complex regulation of this feedback by pyruvate and by some intermediates of the Krebs cycle.

During 2020, the COVID-19 pandemic affected almost 108 individuals. Quite a number of vaccines against COVID-19 were therefore developed, and a few recently received authorization for emergency use. Overall, these vaccines target specific viral proteins by antibodies whose synthesis is directly elicited or indirectly triggered by nucleic acids coding for the desired targets. Among these targets, the receptor binding domain (RBD) of COVID-19 spike protein (SP) does frequently occur in the repertoire of candidate vaccines. However, the immunogenicity of RBD per se is limited by its low molecular mass, and by a structural rearrangement of full-length SP accompanied by the detachment of RBD. Here we show that the RBD of COVID-19 SP can be conveniently produced in Escherichia coli when fused to a fragment of CRM197, a variant of diphtheria toxin currently used for a number of conjugated vaccines. In particular, we show that the CRM197-RBD chimera solubilized from inclusion bodies can be refolded and purified to a state featuring the 5 native disulphide bonds of the parental proteins, the competence in binding angiotensin-converting enzyme 2, and a satisfactory stability at room temperature. Accordingly, our observations provide compulsory information for the development of a candidate vaccine directed against COVID-19.

The DNA polymerase from african swine fever virus (ASFV Pol X), lacking both 8 kDa and thumb domains, is the smallest enzyme featuring competence in DNA extension. Here we show that ASFV Pol X features poor filling activity of DNA gaps consisting of 15 bases, and exerts a more efficient action at the expense of DNA substrates containing a recessed end of equal length. We also show that shortening the recessed end of DNA substrates decreases the rate of DNA elongation catalysed by ASFV Pol X. Finally, by means of stopped-flow experiments we were able to determine that DNA binding is a step responsible for restraining the efficiency of ASFV Pol X catalytic action.

Deinococcus radiodurans is a Gram positive bacterium the capability of which to withstand high doses of ionizing radiations is well known. Physiologically speaking, D. radiodurans is a proteolytic prokaryote able to express and secrete quite a number of proteases, and to use amino acids as an energy source. When considering this, it is surprising that little information is available on the biochemical components responsible for the uptake of peptides in D. radiodurans. Here we report on the purification and characterization of an ABC peptide transporter, isolated from D. radiodurans cells grown in tryptone-glucose-yeast extract (TGY) medium. In particular, we show here that the action of this transporter (denoted DR1571, SwissProt data bank accession number Q9RU24 UF71_DEIRA) is exerted on peptides containing at least 3 amino acids. Further, using tetra-peptides as model systems, we were able to observe that the DR1571 protein does not bind to peptides containing phenylalanine or valine, but associates with high efficiency to tetra-glycine, and with moderate affinity to tetra-peptides containing arginine or aspartate.

Ten manganese(I) tricarbonyl diimine complexes bound to variably functionalised 5-aryl-tetrazolato ligands were prepared, and their photochemical properties were investigated. Upon exposure to light at 365 nm, each complex decomposed to its free diimine and tetrazolato ligands, simultaneously dissociating three CO ligands, as evidenced by changes in the IR spectra of the irradiated complexes over time. The anti-bacterial properties of one of these complexes were tested against Escherichia coli. While the complex displayed no effect on the bacterial growth in the dark, pre-irradiated solutions inhibited bacterial growth. Comparative studies revealed that the antibacterial properties originate from the presence of free 1,10-phenanthroline.

Herein, we describe a new family of tris chelate homoleptic Ru (II) complexes, [Ru(N^N)3]2+, where the role of the diimine-type ligands (N^N) was fulfilled by 2-pyridyl (PTZ) or 2-quinolyl tetrazole (QTZ) derivatives decorated with various alkyl substituents at the N-2 position of the tetrazole ring. The new Ru (II) complexes with general formula [Ru (PTZ-R)3]2+ and [Ru (QTZ-R)3]2+, were obtained as mixtures of facial (fac) and meridional (mer) isomers, as suggested by NMR (1H, 13C) experiments, and confirmed in the case of mer-[Ru (QTZ-Me)3]2+, by X-ray crystallography. The photophysical behavior of the tetrazole-based [Ru(N^N)3]2+ type species was investigated by UV–vis absorption spectroscopy, providing trends typical of polypyridyl Ru (II) complexes. The new homoleptic complexes fac/mer-[Ru (PTZ-R)3]2+ and fac/mer-[Ru (QTZ-R)3]2+ have been assessed for any eventual antimicrobial activity towards two different bacteria such as Gram-negative Escherichia coli and Gram-positive Deinococcus radiodurans. Whereas being inactive toward E. coli, the response of agar disks diffusion tests suggested that some of the new fac/mer Ru (II) complexes could inhibit the growth of D. radiodurans. This effect was further investigated by determining the growth kinetics in liquid medium of D. radiodurans exposed to the fac/mer-[Ru (PTZ-R)3]2+ and fac/mer-[Ru (QTZ-R)3]2+ complexes at different concentrations. The outcome of these experiments highlighted that the turn-on of the growth inhibitory effect took place as the linear hexyl chain was appended to the PTZ or QTZ scaffold, suggesting also how the inhibitory activity appeared more pronouncedly exerted by the facial isomers fac-[Ru (PTZ-Hex)3]2+ and fac-[Ru (QTZ-Hex)3]2+ (MIC = ca. 3.0 μg/ml) with respect to the corresponding meridional isomers (MIC = ca. 6.0 μg/ml).