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Hemoglobin (Hb) plays its vital role through structural and functional properties evolutionarily optimized to work within red blood cells, i.e., the tetrameric assembly, well-defined oxygen affinity, positive cooperativity, and heterotropic allosteric regulation by protons, chloride and 2,3-diphosphoglycerate. Outside red blood cells, the Hb tetramer dissociates into dimers, which exhibit high oxygen affinity and neither cooperativity nor allosteric regulation. They are prone to extravasate, thus scavenging endothelial NO and causing hypertension, and cause nephrotoxicity. In addition, they are more prone to autoxidation, generating radicals. The need to overcome the adverse effects associated with cell-free Hb has always been a major hurdle in the development of substitutes of allogeneic blood transfusions for all clinical situations where blood is unavailable or cannot be used due to, for example, religious objections. This class of therapeutics, indicated as hemoglobin-based oxygen carriers (HBOCs), is formed by genetically and/or chemically modified Hbs. Many efforts were devoted to the exploitation of the wealth of biochemical and biophysical information available on Hb structure, function, and dynamics to design safe HBOCs, overcoming the negative effects of free plasma Hb. Unfortunately, so far, no HBOC has been approved by FDA and EMA, except for compassionate use. However, the unmet clinical needs that triggered intensive investigations more than fifty years ago are still awaiting an answer. Recently, HBOCs “repositioning” has led to their successful application in organ perfusion fluids.

Functionalized antibodies, protein subs titution, supplementation therapies, and enzyme-based therapeutics are protein-based approaches for the treatment and cure of human genetic and acquired diseases. Gene editing and RNA-based therapeutics, including siR NAs, microRNAs, and antisense oligonuc leotides (ASOs), target the ma- chinery that produces disease-associat ed proteins. Protein-based and RNA-based products are collectively referred to as biologics. This Thematic Issue opens a novel specialized s ection of Current Medicinal Chemistry focused on targeted biologics at various stages of discovery, preclinical and clinical development. We hope it might encourage the advancement of this field by stimulating researchers to share their results. Within this issue, readers will find papers discussing th e use of replacement proteins therapeutics, with two ex- amples provided. Alpha-1-antitrypsin and C-1 inhibitor (or C1-esterase inhibitor) both belong to the family of ser- pins and are indicated for the treatment of conditions that result from pat hologically low levels or non-functional variants. Alpha-1-antitrypsin is a small protein that inhib its the catalytic action of el astase, a proteolytic enzyme. When the action of elastase is not properly counterbalan ced by alpha-1-antitrypsin in the lung, pulmonary emphy- sema develops. Alpha-1-antitrypsin was approved more than thirty years ago and is used for the treatment of Alpha- 1-antitrypsin deficiency. The paper by Bianchera et al. [1] reports on issues associated with alpha-1-antitrypsin pro- duction from either animal sources or heterologous expre ssion, focusing on existing and novel protein formulations and delivery strategies. The C-1 inhibitor is involved in a re gulatory network of comple ment, contact, coagulation, fibrinolytic systems and functions as an anti-inflammatory agent in circulation. C-1 inhibitor is used as a prophylactic or acute treatment in Hereditary Angioedema (HAE), which is a rare genetic disease. The paper by Karnaukhova et al. [4] provides an overview of the biochemical properties of C-1 inhibitor, its role in HAE, recent progress in therapeutic strategies for this disease treatment, as well as potential applications fo r sepsis, endotoxin shock, antibody-mediated rejection fol- lowing kidney transplantation, and severe systemic abnormalities related to COVID-19. During the Vietnam War in the late 1960s, the United States started searching for blood substitutes, such as mod- ified hemoglobin or perfluorocarbon solutions, to replace transfusions when blood was unavailable. Since then, many attempts have been carried out, including the devel opment of chemically and/or genetically modified hemo- globins designed to mimic red blood cells. The paper by Sakai et al. [7] summarizes the intense activities carried out predominantly in Japan towards th e development of hemoglobi n-containing liposomes. Issues associated with lipids purity, hemoglobin purification, and stability towards oxidation are presented and discussed. There are many pathophysiologic aberrati ons associated with genetic variants of hemoglobin, often caused by single amino acid substitutions. One such example is the single amino acid Glu-Val substitution at position 6 of the beta chains, leading to sickle cell disease. Hydroxyurea is a mainstay of sickle cell disease therapy to increase the percentage of fetal hemoglobin. Novel marketed therap eutics are based on compounds either interacting directly with hemoglobin to modulate oxygen binding or altering water-controlling pump systems. Alternative and more recently proposed approaches are based on hematopoietic st em cell transplantation and gene therapy platforms. These approaches are reviewed by Garg et al. [3] which discuss advances in precision therapy for sickle cell disease and the preclinical and clinical advances in autologous hematopoietic stem cell gene therapy. Breathing is a fundamental function for all living systems. It is made possible in both animals and humans by the presence of pulmonary surfactant, a complex mixture of lipids and proteins secreted into the alveolar lumen. Its role is the maintenance of lung homeostasis to avoid alveolar collapse. In addition, pulmonary surfactant provides a bar- rier against inhaled pathogens. An insufficient amount of su rfactant or its functional inac tivation is associated with lung pathologies, including neonatal resp iratory distress syndrome. Pioselli et al. [6] reviewed the current state of pulmonary surfactant mimetics in development for replacement therapy. Presently, there are lifesaving products in this class approved for worldwide use in premature infants. Because novel proteins being studied in developmental stages are typically extracted from porcine sources, the pr oduction strategies for tailored surfactant proteins by re- combinant technologies are being refined. Further, pulmonary surfactants ar e attractive candidates for synergistic and carrier molecules that enhance the treatment of a variety of respiratory diseases. Enzymes are biomolecules that catalyze an impressive variety of chemical reactions. A key feature of an enzyme is its specificity for specific substrates. Therefore, it is not surprising that enzymes have been attractive for carrying out selective therapeutic actions. There are two main medicinal strategies based on enzymes: i) supplementation and ii) replacement. Cioni et al. [2] focused on a range of disorders wher e the supplementation of enzymes to patients provides the basis for therapeutic interventions, including clotting disorders, cystic fibrosis, lactose intolerance, col- lagen-based disorders, and cancers. Enzymes for therapeutic indications are increasing as a result of advancements in genetic engineering techniques. Typi cal examples are asparaginase and methionine gamma-lyase, used in cancer therapy to degrade either asparagine or methionine, respec tively. The rationale is based on the vital need for these amino acids by cancer cells. Depletion of these amino acids in the cellular environment slows or prevents cancer cell growth, potentiating the efficacy of traditional chemotherapies. Because enzymes are very "fragile" biomole- cules, novel strategies are being employed to optimize their stability and delivery. Enzyme replacement therapy is also the focus of the paper by Marchetti et al. [5]. Genetic deficiencies of a specific enzyme can lead to the accumu- lation of its substrates and toxic effect s. For example, Gaucher and Fabry diseases are the first rare genetic lysoso- mal storage disorders for the treatment of which enzymes were successfully delivered. In these examples, enzymes were initially obtained from animal sources, then produced using recombinant technology with chemical modifica- tions or genetic variants to enhance and extend their activity. This first issue of the Targeted Biologics represents a small sampling of successful applications of biologics. We are confident that Current Medicinal Chemistry readers will find these examples intellectually stimulating and allow the journal to advance exciting concepts in Biologics for targeted therapy.

Methionine is an essential amino acid involved in the formation of polyamines and a precursor metabolite for DNA and protein methylation. The dependence of cancer cells on methionine has triggered extensive investigations aimed at its targeting for cancer therapy, including the exploitation as a therapeutic tool of methionine γ-lyase (MGL), a bacterial enzyme that degrades methionine, capable of inhibiting cancer cells growth due to methionine starvation. We have exploited the high-resolution power of mass spectrometry to compare the effects of reduced availability of the methyl donor SAM, induced by MGL treatment, on the post-translational modifications of the histone tails in normal Hs27 and cancer HT-29 cells. In the absence of MGL, our analysis detected a three-fold higher relative abundance of trimethylated K25 of H1.4 in HT-29 than Hs27 cells, and a complex pattern of methylated, unmethylated and acetylated peptides in H2 and H3.3. In the presence of MGL, in HT-29, the peptide H2A1_4_11 is predominantly unmodified with mono-methylated K5 increasing upon treatment, whereas in Hs27 cells, H2A1_4_11 is monomethylated at K5 and K9 with these marks decreasing upon treatment. The time dependence of the effects of MGL-mediated methionine depletion on PTMs of histone variants in HT-29 cancer cells was also monitored. Overall, our present data on histone variants H1, H2A, H2B as well as H3.3 integrated with our previous studies on histones H3 and H4, shed light on the epigenetic modifications associated with methionine starvation and associated cancer cell death.

Together with their undeniable advantages, the new technologies of the Fintech Revolution bring new risks. Some of these risks are already known but have taken on a new form; some are entirely new. Among the latter, one of the most relevant concerns the opacity of artificial intelligence (AI).

Up to 40% of patients with inflammatory bowel disease present with psychosocial disturbances. We previously identified a gut vascular barrier that controls the dissemination of bacteria from the intestine to the liver. Here, we describe a vascular barrier in the brain choroid plexus (PVB) that is modulated in response to intestinal inflammation through bacteria-derived lipopolysaccharide. The inflammatory response induces PVB closure after gut vascular barrier opening by the up-regulation of the wingless-type, catenin-beta 1 (Wnt/β-catenin) signaling pathway, rendering it inaccessible to large molecules. In a model of genetically driven closure of choroid plexus endothelial cells, we observed a deficit in short-term memory and anxiety-like behavior, suggesting that PVB closure may correlate with mental deficits. Inflammatory bowel disease–related mental symptoms may thus be the consequence of a deregulated gut–brain vascular axis.

Methionine is an essential amino acid used, beyond protein synthesis, for polyamine formation and DNA/RNA/protein methylation. Cancer cells require particularly high methionine supply for their homeostasis. A successful approach for decreasing methionine concentration is based on the systemic delivery of methionine γ-lyase (MGL), with in vitro and in vivo studies demonstrating its efficacy in cancer therapy. However, the mechanisms explaining how cancer cells suffer from the absence of methionine more significantly than non-malignant cells are still unclear. We analyzed the outcome of the human colorectal adenocarcinoma cancer cell line HT29 to the exposure of MGL for up to 72 h by monitoring cell viability, proteome expression, histone post-translational modifications, and presence of spurious transcription. The rationale of this study was to verify whether reduced methionine supply would affect chromatin decondensation by changing the levels of histone methylation and therefore increasing genomic instability. MGL treatment showed a time-dependent cytotoxic effect on HT29 cancer cells, with an IC50 of 30 µg/ml, while Hs27 normal cells were less affected, with an IC50 of >460 µg/ml. Although the levels of total histone methylation were not altered, a loss of the silencing histone mark H3K9me2 was observed, as well as a decrease in H4K20me3. Since H3K9me2/3 decorate repetitive DNA elements, we proved by qRT-PCR that MGL treatment leads to an increased expression of major satellite units. Our data indicate that selected histone methylation marks may play major roles in the mechanism of methionine starvation in cancer cells, proving that MGL treatment directly impacts chromatin homeostasis.

Background and Aims The SARS-CoV-2 pandemic has overwhelmed the treatment capacity of the health care systems during the highest viral diffusion rate. Patients reaching the emergency department had to be either hospitalized (inpatients) or discharged (outpatients). Still, the decision was taken based on the individual assessment of the actual clinical condition, without specific biomarkers to predict future improvement or deterioration, and discharged patients often returned to the hospital for aggravation of their condition. Here, we have developed a new combined approach of omics to identify factors that could distinguish coronavirus disease 19 (COVID-19) inpatients from outpatients. Methods Saliva and blood samples were collected over the course of two observational cohort studies. By using machine learning approaches, we compared salivary metabolome of 50 COVID-19 patients with that of 270 healthy individuals having previously been exposed or not to SARS-CoV-2. We then correlated the salivary metabolites that allowed separating COVID-19 inpatients from outpatients with serum biomarkers and salivary microbiota taxa differentially represented in the two groups of patients. Results We identified nine salivary metabolites that allowed assessing the need of hospitalization. When combined with serum biomarkers, just two salivary metabolites (myo-inositol and 2-pyrrolidineacetic acid) and one serum protein, chitinase 3-like-1 (CHI3L1), were sufficient to separate inpatients from outpatients completely and correlated with modulated microbiota taxa. In particular, we found Corynebacterium 1 to be overrepresented in inpatients, whereas Actinomycetaceae F0332, Candidatus Saccharimonas, and Haemophilus were all underrepresented in the hospitalized population. Conclusion This is a proof of concept that a combined omic analysis can be used to stratify patients independently from COVID-19.

State of the art vehicle dynamics control systems do not exploit tire road forces information, even though the vehicle behaviour is ultimately determined by the tire road interaction. Recent technological improvements allow to accurately measure and estimate these variables, making it possible to introduce such knowledge inside a control system. In this paper, a vehicle dynamics control architecture based on a direct longitudinal tire force feedback is proposed. The scheme is made by a nested architecture composed by an outer Model Predictive Control algorithm, written in spatial coordinates, and an inner longitudinal force feedback controller. The latter is composed by four classical Proportional-Integral controllers in anti-windup configuration, endowed with a suitably designed gain switching logic to cope with possible unfeasible references provided by the outer loop, avoiding instability. The proposed scheme is tested in simulation in a challenging scenario where the tracking of a spiral path on a slippery surface and the timing performance are handled simultaneously by the controller. The performance is compared with that of an inner slip-based controller, sharing the same outer Model Predictive Control loop. The results show comparable performance in presence of unfeasible force references, while higher robustness is achieved with respect to friction curve uncertainties.

Hemoglobin (Hb)-based oxygen carriers (HBOC) are modified extracellular proteins, designed to replace or augment the oxygen-carrying capacity of erythrocytes. However, clinical results have generally been disappointing due to adverse side effects, in part linked to the intrinsic oxidative toxicity of Hb. Previously a redox-active tyrosine residue was engineered into the Hb β subunit (βF41Y) to facilitate electron transfer between endogenous antioxidants such as ascorbate and the oxidative ferryl heme species, converting the highly oxidizing ferryl species into the less reactive ferric (met) form. We inserted different single tyrosine mutations into the α and β subunits of Hb to determine if this effect of βF41Y was unique. Every mutation that was inserted within electron transfer range of the protein surface and the heme increased the rate of ferryl reduction. However, surprisingly, three of the mutations (βT84Y, αL91Y and βF85Y) also increased the rate of ascorbate reduction of ferric(met) Hb to ferrous(oxy) Hb. The rate enhancement was most evident at ascorbate concentrations equivalent to that found in plasma (< 100 μM), suggesting that it might be of benefit in decreasing oxidative stress in vivo. The most promising mutant (βT84Y) was stable with no increase in autoxidation or heme loss. A decrease in membrane damage following Hb addition to HEK cells correlated with the ability of βT84Y to maintain the protein in its oxygenated form. When PEGylated and injected into mice, βT84Y was shown to have an increased vascular half time compared to wild type PEGylated Hb. βT84Y represents a new class of mutations with the ability to enhance reduction of both ferryl and ferric Hb, and thus has potential to decrease adverse side effects as one component of a final HBOC product., Graphical abstract fx1, Highlights • Cell free Hemoglobin Based Oxygen Carriers can deliver oxygen to tissue. • Oxidation of ferrous hemoglobin to ferric and ferryl leads to oxidative toxicity. • Insertion of tyrosine residues enhances reduction of ferryl to ferric hemoglobin. • Some mutants (e.g. βT84Y) also enhance ferric to ferrous reduction by ascorbate. • βT84Y decreases cellular oxidative stress and enhances vascular retention in vivo.

Over the past decade, infectious agents, including bacteria, have come under scrutiny for contributing to almost 16% of all tumors. Indeed, emerging data report a microbial presence in many cancer types, with breast cancer accumulating the most diverse bacterial population. Pathogen defense mechanisms, such as CHI3L1, contribute to bacterial elimination; however, CHI3L1 is overexpressed in several cancer types and correlates with poor prognosis and shorter survival. However, the connection between intra-tumoral bacteria and CHI3L1 and how they contribute to cancer growth has not been fully characterized.To investigate if microbiota and CHI3L1 are connected, female BALB/c mice harboring 4T1-Luc cells were exposed to a broad-spectrum antibiotic mix to deplete the microbiota and to evaluate tumor growth and circulating CHI3L1 levels. 9 and 14 days after tumor inoculation, feces were collected and bacterial DNA isolated for 16S rRNA sequencing, and tumors were plated to identify intratumoral bacteria. To assess whether a specific bacterial strain could modulate CHI3L1 expression, we incubated 4T1 cells with supernatants from isolated bacteria, which contain metabolites and extracellular factors. Isolated bacteria that could upregulate CHI3L1 expression were then used to infect 4T1 cells prior to subcutaneous injection in the mammary fat pad of BALB/c mice to evaluate if bacterial infection changes tumor growth rate.In the 4T1 mouse breast cancer model, a broad-spectrum antibiotic treatment was able to: deplete host microbiota, abrogate tumor growth, and reduce CHI3L1 levels in the sera and in CD45+ immune infiltrating cells in tumor and colon. When 4T1 cells were exposed to bacterial supernatants in vitro, we observed that CHI3L1 was upregulated by a specific strain of E. coli. To simulate the effect of intra-tumoral microbiota, we infected 4T1 cells with either the isolated E. coli or S. aureus (which did not induce any CHI3L1 modulation in vitro) prior to subcutaneous injection. Strikingly, E. coli infection increased tumor progression with respect to the control S. aureus infection (p Citation Format: Alessandro Mauro Mozzarelli, Sara Carloni, Valentina Ferrrari, Silvia Giugliano, Luca Tiraboschi, Antonino Lo Cascio, Giulia Fornasa, Daniele Braga, Davide Golzato, Nicola Segata, Giuseppe Penna, Maria Rescigno. Microbiota mediated CHI3L1 expression as a novel candidate biomarker for breast cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3059.

Background Wrist fractures are a common occurrence affecting patients of all ages. Wrist fracture patients often develop oedema and the presence of cast increases the risk. The use of an adhesive elastic tape is indicated for the oedema control, but there is no definitive evidence of its effectiveness. This study aims to evaluate the effectiveness of the tape in the hand oedema control of wrist fracture patients with forearm cast. Methods Randomized controlled trial with blinded data processing. We will apply the tape to the intervention group after the plaster application, the control group will receive the standard treatment. We will evaluate the circumference difference between baseline (T0) and the 7-day follow-up (T1) of both the thumb and of the remaining 4 fingers merged together. Moreover, re-attendance data to the Orthopedic Emergency Room due to “intolerance to the plaster cast” will be checked. Sample size calculations resulted in a total of 220 (110 per group). Ethical approval for the study has been obtained. Discussion We aim to demonstrate that the use of tape improves the tolerability of the cast by reducing the oedema formation, the feeling of constriction and pain. Trial registration ClinicalTrial.gov Identifier: NCT04683887.

Antibacterial adjuvants are of great significance, since they allow the therapeutic dose of conventional antibiotics to be lowered and reduce the insurgence of antibiotic resistance. Herein, we report that an

Many bacteria and actinomycetales use L-cysteine biosynthesis to increase their tolerance to antibacterial treatment and establish a long-lasting infection. In turn, this might lead to the onset of antimicrobial resistance that currently represents one of the most menacing threats to public health worldwide. The biosynthetic machinery required to synthesise L-cysteine is absent in mammals, therefore, its exploitation as a drug target is particularly promising. In this article, we report a series of inhibitors of Salmonella thyphimurium serine acetyltransferase (SAT), the enzyme that catalyzes the rate-limiting step of L-cysteine biosynthesis. The development of such inhibitors started with the virtual screening of an in-house library of compounds that led to the selection of seven structurally unrelated hit derivatives. A set of molecules structurally related to hit compound 5, coming either from the original library or from medicinal chemistry efforts, were tested to determine a preliminary structure–activity relationship and, especially, to improve the inhibitory potency of the derivatives, that was indeed ameliorated by several folds compared to hit compound 5 Despite these progresses, at this stage, the most promising compound failed to interfere with bacterial growth when tested on a Gram-negative model organism, anticipating the need for further research efforts.

Cysteine is the main precursor of sulfur-containing biological molecules in bacteria and contributes to the control of the cell redox state. Hence, this amino acid plays an essential role in microbial survival and pathogenicity and the reductive sulfate assimilation pathway is considered a promising target for the development of new antibacterials. Serine acetyltransferase (SAT) and O-acetylserine sulfhydrylase (OASS-A), the enzymes catalyzing the last two steps of cysteine biosynthesis, engage in the formation of the cysteine synthase (CS) complex. The interaction between SAT and OASS-A finely tunes cysteine homeostasis, and the development of inhibitors targeting either protein–protein interaction or the single enzymes represents an attractive strategy to undermine bacterial viability. Given the peculiar mode of interaction between SAT and OASS-A, which exploits the insertion of SAT C-terminal sequence into OASS-A active site, we tested whether a recently developed competitive inhibitor of OASS-A exhibited any effect on the CS stability. Through surface plasmon resonance spectroscopy, we (i) determined the equilibrium constant for the Salmonella Typhimurium CS complex formation and (ii) demonstrated that the inhibitor targeting OASS-A active site affects CS complex formation. For comparison, the Escherichia coli CS complex was also investigated, with the aim of testing the potential broad-spectrum activity of the candidate antimicrobial compound.

Genome instability is a condition characterized by the accumulation of genetic alterations and is a hallmark of cancer cells. To uncover new genes and cellular pathways affecting endogenous DNA damage and genome integrity, we exploited a Synthetic Genetic Array (SGA)-based screen in yeast. Among the positive genes, we identified VID22, reported to be involved in DNA double-strand break repair. vid22Δ cells exhibit increased levels of endogenous DNA damage, chronic DNA damage response activation and accumulate DNA aberrations in sequences displaying high probabilities of forming G-quadruplexes (G4-DNA). If not resolved, these DNA secondary structures can block the progression of both DNA and RNA polymerases and correlate with chromosome fragile sites. Vid22 binds to and protects DNA at G4-containing regions both in vitro and in vivo. Loss of VID22 causes an increase in gross chromosomal rearrangement (GCR) events dependent on G-quadruplex forming sequences. Moreover, the absence of Vid22 causes defects in the correct maintenance of G4-DNA rich elements, such as telomeres and mtDNA, and hypersensitivity to the G4-stabilizing ligand TMPyP4. We thus propose that Vid22 is directly involved in genome integrity maintenance as a novel regulator of G4 metabolism., Associazione Italiana per la Ricerca sul Cancro (AIRC) [15631, 21806 to M.M.F.]; MIUR [PRIN 2015-2015SJLMB9; PRIN 2017-2017KSZZJW to M.M.F.]; Telethon [GGP15227 to M.M.F.]; F.L. was supported by the University of Milano: ‘‘Piano di Sviluppo dell’Ateneo per la Ricerca. Linea B: Supporto per i Giovani Ricercatori’’; M.C.B. was supported by Fondazione Veronesi; Research at the laboratory of A.A. was funded by the Spanish Ministry of Economy and Competitiveness [BFU2016-75058-P]; B.G.G. was funded by the Spanish Association Against Cancer; MIUR [PRIN2017-2017Z55KC to T.B.]; M.C., D.S.H. are supported by MIUR [PRIN 2017] and CNRbiomics [PIR01_00017]; H2020 Projects ELIXIR-EXCELERATE, EOSC-Life, EOSC-Pillar and Elixir-IIB; G.W.B. was supported by the Canadian Institutes of Health Research[FDN-159913]. Funding for open access charge: Associazione Italiana per la Ricerca sul Cancro (AIRC) [21806].

Human serine racemase (hSR) catalyzes the biosynthesis of D-serine, an obligatory co-agonist of the NMDA receptors. It was previously found that the reversible S-nitrosylation of Cys113 reduces hSR activity. Here, we show by site-directed mutagenesis, fluorescence spectroscopy, mass spectrometry, and molecular dynamics that S-nitrosylation stabilizes an open, less-active conformation of the enzyme. The reaction of hSR with either NO or nitroso donors is conformation-dependent and occurs only in the conformation stabilized by the allosteric effector ATP, in which the e-amino group of Lys114 acts as a base towards the thiol group of Cys113. In the closed conformation stabilized by glycine - an active-site ligand of hSR - the side chain of Lys114 moves away from that of Cys113, while the carboxyl side-chain group of Asp318 moves significantly closer, increasing the thiol pKa and preventing the reaction. We conclude that ATP binding, glycine binding, and S-nitrosylation constitute a three-way regulation mechanism for the tight control of hSR activity. We also show that Cys113 undergoes H2 O2 -mediated oxidation, with loss of enzyme activity, a reaction also dependent on hSR conformation.

Murine serine racemase (SR), the enzyme responsible for the biosynthesis of the neuromodulator d-serine, was reported to form a complex with glyceraldehyde 3-phosphate dehydrogenase (GAPDH), resulting in SR inhibition. In this work, we investigated the interaction between the two human orthologues. We were not able to observe neither the inhibition nor the formation of the SR-GAPDH complex. Rather, hSR is inhibited by the hGAPDH substrate glyceraldehyde 3-phosphate (G3P) in a time- and concentration-dependent fashion, likely through a covalent reaction of the aldehyde functional group. The inhibition was similar for the two G3P enantiomers but it was not observed for structurally similar aldehydes. We ruled out a mechanism of inhibition based on the competition with either pyridoxal phosphate (PLP) - described for other PLP-dependent enzymes when incubated with small aldehydes - or ATP. Nevertheless, the inhibition time course was affected by the presence of hSR allosteric and orthosteric ligands, suggesting a conformation-dependence of the reaction.

Catalysis makes chemical and biochemical reactions kinetically accessible. From a technological point of view, organic, inorganic, and biochemical catalysis is relevant for several applications, from industrial synthesis to biomedical, material, and food sciences. A heterogeneous catalyst, i.e., a catalyst confined in a different phase with respect to the reagents’ phase, requires either its physical confinement in an immobilization matrix or its physical adsorption on a surface. In this review, we will focus on the immobilization of biological catalysts, i.e., enzymes, by comparing hard and soft immobilization matrices and their effect on the modulation of the catalysts’ function. Indeed, unlike smaller molecules, the catalytic activity of protein catalysts depends on their structure, conformation, local environment, and dynamics, properties that can be strongly affected by the immobilization matrices, which, therefore, not only provide physical confinement, but also modulate catalysis.

[Image: see text] Covalent inhibitors of PfGAPDH characterized by a 3-bromoisoxazoline warhead were developed, and their mode of interaction with the target enzyme was interpreted by means of molecular modeling studies: some of them displayed a submicromolar antiplasmodial activity against both chloroquine sensitive and resistant strains of Plasmodium falciparum, with good selectivity indices.