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We investigate the inhibition mechanism between pomotrelvir and the SARS-CoV-2 main protease using molecular mechanics and quantum mechanics/molecular mechanics simulations. Alchemical transformations where each Pi group of pomotrelvir was transformed into its counterpart in nirmatrelvir were performed to unravel the individual contribution of each group to the binding and reaction processes. We have shown that while a γ-lactam ring is preferred at position P1, a δ-lactam ring is a good alternative for the design of inhibitors for variants presenting mutations at position 166. For the P2 position, tertiary amines are preferred with respect to secondary amines. Flexible side chains at the P2 position can disrupt the preorganization of the active site, favouring the exploration of non-reactive conformations. The substitution of the P2 group of pomotrelvir by that of nirmatrelvir resulted in a compound, named as C2, that presents a better binding free energy and a higher population of reactive conformations in the Michaelis complex. Analysis of the chemical reaction to form the covalent complex has shown a similar reaction mechanism and activation free energies for pomotrelvir, nirmatrelvir and C2. We hope that these findings could be useful to design better inhibitors to fight present and future variants of the SARS-CoV-2 virus., (© 2024 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

We present in this work the emle-engine package (https://github.com/chemle/emle-engine)─the implementation of a new machine learning embedding scheme for hybrid machine learning potential/molecular-mechanics (ML/MM) dynamics simulations. The package is based on an embedding scheme that uses a physics-based model of the electronic density and induction with a handful of tunable parameters derived from in vacuo properties of the subsystem to be embedded. This scheme is completely independent of the in vacuo potential and requires only the positions of the atoms of the machine learning subsystem and the positions and partial charges of the molecular mechanics environment. These characteristics allow emle-engine to be employed in existing QM/MM software. We demonstrate that the implemented electrostatic machine learning embedding scheme (named EMLE) is stable in enhanced sampling molecular dynamics simulations. Through the calculation of free energy surfaces of alanine dipeptide in water with two different ML options for the in vacuo potential and three embedding models, we test the performance of EMLE. When compared to the reference DFT/MM surface, the EMLE embedding is clearly superior to the MM one based on fixed partial charges. The configurational dependence of the electronic density and the inclusion of the induction energy introduced by the EMLE model leads to a systematic reduction in the average error of the free energy surface when compared to MM embedding. By enabling the usage of EMLE embedding in practical ML/MM simulations, emle-engine will make it possible to accurately model systems and processes that feature significant variations in the charge distribution of the ML subsystem and/or the interacting environment.

Ubiquitin-specific protease 7 (USP7) is a deubiquitinase enzyme that plays a critical role in regulating various cellular processes by cleaving ubiquitin molecules from target proteins. The C-terminal loop (CTL) motif is a specific region at the C-terminal end of the USP7 enzyme. Recent experiments suggest that the CTL motif plays a role in USP7's catalytic activity by contributing to the enzyme's structural stability, substrate recognition, and catalytic efficiency. The objective of this work is to elucidate these roles through the utilization of computational methods for molecular simulations. For this, we conducted extensive molecular dynamics (MD) simulations to investigate the conformational dynamics and protein-protein interactions within the USP7 enzyme-substrate complex with the substrate consisting of the ubiquitin tagged with the fluorescent label rhodamine 110-gly (Ub-Rho). Our results demonstrate that the CTL motif plays a crucial role in stabilizing the Ubl domains' conformation and augmenting the stability of active conformations within the enzyme-substrate complex. Conversely, the absence of the CTL motif results in increased flexibility and variability in Ubl domains' motion, leading to a reduced percentage of active conformations. Furthermore, our analysis of protein-protein interactions highlights the significance of the CTL motif in anchoring the Ubl45 domains to the catalytic domain (CD), thereby facilitating stable interactions with the substrate. Overall, our findings provide valuable insights into the conformational dynamics and protein-protein interactions inherent in the USP7 enzyme-substrate complex. These insights shed light on some mechanistic details of USP7 concerning the substrate's recognition before its catalytic action.

The formation of an amide bond is an essential step in the synthesis of materials and drugs, and in the assembly of amino acids to form peptides. The mechanism of this reaction has been studied extensively, in particular to understand how it can be catalyzed, but a representation capable of explaining all the experimental data is still lacking. Numerical simulation should provide the necessary molecular description, but the solvent involvement poses a number of challenges. Here, we combine the efficiency and accuracy of neural network potential-based reactive molecular dynamics with the extensive and unbiased exploration of reaction pathways provided by transition path sampling. Using microsecond-scale simulations at the density functional theory level, we show that this method reveals the presence of two competing distinct mechanisms for peptide bond formation between alanine esters in aqueous solution. We describe how both reaction pathways, via a general base catalysis mechanism and via direct cleavage of the tetrahedral intermediate respectively, change with pH. This result contrasts with the conventional mechanism involving a single pathway in which only the barrier heights are affected by pH. We show that this new proposal involving two competing mechanisms is consistent with the experimental data, and we discuss the implications for peptide bond formation under prebiotic conditions and in the ribosome. Our work shows that integrating deep potential molecular dynamics with path sampling provides a powerful approach for exploring complex chemical mechanisms.

Fundamental movement skills (FMS), considered as building blocks of movement, have received growing interest due to their significant impact on both present and future health. FMS are categorized into locomotor, object control and stability skills. While there has been extensive research on assessing the proficiency and reliability of locomotor and object control skills, stability skills have received comparatively less attention. For this reason, this study aimed to assess the test-retest, intrarater and interrater reliability of five stability skills included in the Alfamov app. The performance of eighty-four healthy primary school children (60.8% girls), aged 6 to 12 years (mean ± standard deviation of 8.7 ± 1.8 years), in five stability skills was evaluated and scored by four raters, including two experts and two novices. The Alfamov tool, integrating various process-oriented tests, was used for the assessment. Reliability analyses were conducted through the computation of the intraclass correlation coefficient (ICC) along with the corresponding 95% confidence intervals. Good-to-excellent intrarater reliability, excellent interrater reliability and moderate-to-good reliability in the test-retest were achieved. The results proved that Alfamov is a robust test for evaluating stability skills and can be suitable for use by different professionals with less experience in assessing children's motor competence.

Protein loop dynamics have recently been recognized as central to enzymatic activity, specificity and stability. However, the factors controlling loop opening and closing kinetics have remained elusive. Here, we combine molecular dynamics simulations with string-method determination of complex reaction coordinates to elucidate the molecular mechanism and rate-limiting step for WPD-loop dynamics in the PTP1B enzyme. While protein conformational dynamics is often represented as diffusive motion hindered by solvent viscosity and internal friction, we demonstrate that loop opening and closing is activated. It is governed by torsional rearrangement around a single loop peptide group and by significant friction caused by backbone adjustments, which can dynamically trap the loop. Considering both torsional barrier and time-dependent friction, our calculated rate constants exhibit very good agreement with experimental measurements, reproducing the change in loop opening kinetics between proteins. Furthermore, we demonstrate the applicability of our results to other enzymatic loops, including the M20 DHFR loop, thereby offering prospects for loop engineering potentially leading to enhanced designs., (© 2024. The Author(s).)

Enzyme design faces challenges related to the implementation of the basic principles that govern the catalytic activity in natural enzymes. In this work, we revisit basic electrostatic concepts that have been shown to explain the origin of enzymatic efficiency like preorganization and reorganization. Using magnitudes such as the electrostatic potential and the electric field generated by the protein, we explain how these concepts work in different enzymes and how they can be used to rationalize the consequences of point mutations. We also discuss examples of protein design in which electrostatic effects have been implemented. For the near future, molecular simulations, coupled with the use of machine learning methods, can be used to implement electrostatics as a guiding principle for enzyme designs.

We computationally study the Zika NS3 helicase, a biological motor, using ATP hydrolysis energy for nucleic acid remodeling. Through molecular mechanics and hybrid quantum mechanics/molecular mechanics simulations, we explore the conformational landscape of motif V, a conserved loop connecting the active sites for ATP hydrolysis and nucleic acid binding. ATP hydrolysis, initiated by a meta-phosphate group formation, involves the nucleophilic attack of a water molecule activated by Glu286 proton abstraction. Motif V hydrogen bonds to this water via the Gly415 backbone NH group, assisting hydrolysis. Posthydrolysis, free energy is released when the inorganic phosphate moves away from the coordination shell of the magnesium ion, inducing a significant shift in the conformational landscape of motif V to establish a hydrogen bond between the Gly415 NH group and Glu285. According to our simulations, the Zika NS3 helicase acts as a ratchet biological motor with motif V transitions steered by Gly415's γ-phosphate sensing in the ATPase site.

l-asparaginases catalyze the asparagine hydrolysis to aspartate. These enzymes play an important role in the treatment of acute lymphoblastic leukemia because these cells are unable to produce their own asparagine. Due to the immunogenic response and various side effects of enzymes of bacterial origin, many attempts have been made to replace these enzymes with mammalian enzymes such as human asparaginase type III (hASNaseIII). This study investigates the reaction mechanism of hASNaseIII through molecular dynamics simulations, quantum mechanics/molecular mechanics methods, and free energy calculations. Our simulations reveal that the dimeric form of the enzyme plays a vital role in stabilizing the substrate in the active site, despite the active site residues coming from a single protomer. Protomer-protomer interactions are essential to keep the enzyme in an active conformation. Our study of the reaction mechanism indicates that the self-cleavage process that generates an N-terminal residue (Thr168) is required to activate the enzyme. This residue acts as the nucleophile, attacking the electrophilic carbon of the substrate after a proton transfer from its hydroxyl group to the N-terminal amino group. The reaction mechanism proceeds with the formation of an acyl-enzyme complex and its hydrolysis, which turns out to be the rate-determining step. Our proposal of the enzymatic mechanism sheds light on the role of different active site residues and rationalizes the studies on mutations. The insights provided here about hASNaseIII activity could contribute to the comprehension of the disparities among different ASNases and might even guide the design of new variants with improved properties for acute lymphoblastic leukemia treatment.

Polyethylene terephthalate (PET) is the most abundant polyester plastic, widely used in textiles and packaging, but, unfortunately, it is also one of the most discarded plastics after one use. In the last years, the enzymatic biodegradation of PET has sparked great interest owing to the discovery and subsequent mutation of PETase-like enzymes, able to depolymerize PET. FAST-PETase is one of the best enzymes hitherto proposed to efficiently degrade PET, although the origin of its efficiency is not completely clear. To understand the molecular origin of its enhanced catalytic activity, we have carried out a thorough computational study of PET degradation by the FAST-PETase action by employing classical and hybrid (QM/MM) molecular dynamics (MD) simulations. Our findings show that the rate-limiting reaction step for FAST-PETase corresponds to the acylation stage with an estimated free energy barrier of 12.1 kcal mol -1 , which is significantly smaller than that calculated for PETase (16.5 kcal mol -1 ) and, therefore, supports the enhanced catalytic activity of FAST-PETase. The origin of this enhancement is mainly attributed to the N233K mutation, which, although sited relatively far from the active site, induces a chain folding where the Asp206 of the catalytic triad is located, impeding that this residue sets effective H-bonds with its neighboring residues. This effect makes Asp206 hold a more basic character compared to the wild-type PETase and boosts the interaction with the protonated His237 of the catalytic triad in the transition state of acylation, with the consequent decrease of the catalytic barrier and acceleration of the PET degradation reaction.

Newly growing evidence highlights the essential role that epitranscriptomic marks play in the development of many cancers; however, little is known about the role and implications of altered epitranscriptome deposition in prostate cancer. Here, we show that the transfer RNA N 7 -methylguanosine (m 7 G) transferase METTL1 is highly expressed in primary and advanced prostate tumours. Mechanistically, we find that METTL1 depletion causes the loss of m 7 G tRNA methylation and promotes the biogenesis of a novel class of small non-coding RNAs derived from 5'tRNA fragments. 5'tRNA-derived small RNAs steer translation control to favour the synthesis of key regulators of tumour growth suppression, interferon pathway, and immune effectors. Knockdown of Mettl1 in prostate cancer preclinical models increases intratumoural infiltration of pro-inflammatory immune cells and enhances responses to immunotherapy. Collectively, our findings reveal a therapeutically actionable role of METTL1-directed m 7 G tRNA methylation in cancer cell translation control and tumour biology., (© 2023. The Author(s).)

The circadian clock controls behavior and physiology. Presently, there is clear evidence of a connection between this timing system and cancer development/progression. Moreover, circadian rhythm consideration in the therapeutic action of anticancer drugs can enhance the effectiveness of cancer therapy. Nanosized drug delivery systems (DDS) have been demonstrated to be suitable engineered platforms for drug targeted/sustained release. The investigation of the chronobiology-nanotechnology relationship, i.e., timing DDS performance according to a patient's circadian rhythm, may greatly improve cancer clinical outcomes. In the present work, we synthesized nanosystems based on an octa-arginine (R8)-modified poly(amidoamine) dendrimer conjugated with the anticancer drug paclitaxel (PTX), G4-PTX-R8, and its physicochemical properties were revealed to be appropriate for in vitro delivery. The influence of the circadian rhythm on its cellular internalization efficiency and potential therapeutic effect on human cervical cancer cells (HeLa) was studied. Cell-internalized PTX and caspase activity, as a measure of induced apoptosis, were monitored for six time points. Higher levels of PTX and caspase-3/9 were detected at T8, suggesting that the internalization of G4-PTX-R8 into HeLa cells and apoptosis are time-specific/-regulated phenomena. For a deeper understanding, the clock protein Bmal1-the main regulator of rhythmic activity, was silenced by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology. Bmal1 silencing was revealed to have an impact on both PTX release and caspase activity, evidencing a potential role for circadian rhythm on drug delivery/therapeutic effect mediated by G4-PTX-R8.

Chronic venous disease (CVD) is a common condition that affects the veins in the lower limbs, resulting in a variety of symptoms, such as swelling, pain, and varicose veins (VVs). The plenty hormonal, hemodynamic and mechanical changes occurred in pregnancy make women especially vulnerable to suffer from this condition in this period. Previous works have identified that CVD is associated with an increased inflammatory milieu and significant damage in maternofetal tissues, such as the umbilical cord. However, the inflammatory status of this structure in these patients has not been studied yet. Thus, the aim of the present study was to examine gene and protein expression of a set of inflammatory markers-Allograft inflammatory factor 1 (AIF-1), the proinflammatory cytokines interleukin 12A (IL-12A) and IL-18 and the anti-inflammatory product IL-10-in the umbilical cord of women with CVD during pregnancy (N = 62) and healthy pregnant women (HC; N = 52) by the use of real time qPCR and immunohistochemistry (IHC). Our results demonstrate that the umbilical cord tissue from CVD women exhibit an increased expression of AIF-1, IL-12A and IL-18 along with a decrease in IL-10. Therefore, our study suggests an inflammatory status of this structure related to CVD. Further studies should be conducted to evaluate the expression of other inflammatory markers, as well as to analyze the maternofetal impact of these findings.

Histone acetylation plays a vital role in organizing chromatin, regulating gene expression and controlling the cell cycle. The first histone acetyltransferase to be identified was histone acetyltransferase 1 (HAT1), but it remains one of the least understood acetyltransferases. HAT1 catalyzes the acetylation of newly synthesized H4 and, to a lesser extent, H2A in the cytoplasm. However, 20 min after assembly, histones lose acetylation marks. Moreover, new noncanonical functions have been described for HAT1, revealing its complexity and complicating the understanding of its functions. Recently discovered roles include facilitating the translocation of the H3H4 dimer into the nucleus, increasing the stability of the DNA replication fork, replication-coupled chromatin assembly, coordination of histone production, DNA damage repair, telomeric silencing, epigenetic regulation of nuclear lamina-associated heterochromatin, regulation of the NF-κB response, succinyl transferase activity and mitochondrial protein acetylation. In addition, the functions and expression levels of HAT1 have been linked to many diseases, such as many types of cancer, viral infections (hepatitis B virus, human immunodeficiency virus and viperin synthesis) and inflammatory diseases (chronic obstructive pulmonary disease, atherosclerosis and ischemic stroke). The collective data reveal that HAT1 is a promising therapeutic target, and novel therapeutic approaches, such as RNA interference and the use of aptamers, bisubstrate inhibitors and small-molecule inhibitors, are being evaluated at the preclinical level.

Caspases are cysteine proteases in charge of breaking a peptide bond next to an aspartate residue. Caspases constitute an important family of enzymes involved in cell death and inflammatory processes. A plethora of diseases, including neurological and metabolic diseases and cancer, are associated with the poor regulation of caspase-mediated cell death and inflammation. Human caspase-1 in particular carries out the transformation of the pro-inflammatory cytokine pro-interleukin-1β into its active form, a key process in the inflammatory response and then in many diseases, such as Alzheimer's disease. Despite its importance, the reaction mechanism of caspases has remained elusive. The standard mechanistic proposal valid for other cysteine proteases and that involves the formation of an ion pair in the catalytic dyad is not supported by experimental evidence. Using a combination of classical and hybrid DFT/MM simulations, we propose a reaction mechanism for the human caspase-1 that explains experimental observations, including mutagenesis, kinetic, and structural data. In our mechanistic proposal, the catalytic cysteine, Cys285, is activated after a proton transfer to the amide group of the scissile peptide bond, a process facilitated by hydrogen-bond interactions with Ser339 and His237. The catalytic histidine does not directly participate in any proton transfer during the reaction. After formation of the acylenzyme intermediate, the deacylation step takes place through the activation of a water molecule by the terminal amino group of the peptide fragment formed during the acylation step. The overall activation free energy obtained from our DFT/MM simulations is in excellent agreement with the value derived from the experimental rate constant, 18.7 vs 17.9 kcal·mol -1 , respectively. Simulations of the H237A mutant support our conclusions and agree with the reported reduced activity observed for this caspase-1 variant. We propose that this mechanism can explain the reactivity of all cysteine proteases belonging to the CD clan and that differences with respect to other clans could be related to the larger preference showed by enzymes of the CD clan for charged residues at position P1. This mechanism would avoid the free energy penalty associated with the formation of an ion pair. Finally, our structural description of the reaction process can be useful to assist in the design of inhibitors of caspase-1, a target in the treatment of several human diseases., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

The use of antiviral drugs can promote the appearance of mutations in the target protein that increase the resistance of the virus to the treatment. This is also the case of nirmatrelvir, a covalent inhibitor of the 3CL protease, or main protease, of SARS-CoV-2. In this work we show how the by-residue decomposition of noncovalent interactions established between the drug and the enzyme, in combination with an analysis of naturally occurring mutations, can be used to detect potential mutations in the 3CL protease conferring resistance to nirmatrelvir. We also investigate the consequences of these mutations on the reaction mechanism to form the covalent enzyme-inhibitor complex using QM/MM methods. In particular, we show that the E166V variant of the protease displays smaller binding affinity to nirmatrelvir and larger activation free energy for the formation of the covalent complex, both factors contributing to the observed resistance to the treatment with this drug. The conclusions derived from our work can be used to anticipate the consequences of the introduction of nirmatrelvir in the fitness landscape of the virus and to design new inhibitors adapted to some of the possible resistance mechanisms., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Glycosylation is recognized as a key process for proper megakaryopoiesis and platelet formation. The enzyme uridine diphosphate (UDP)-galactose-4-epimerase, encoded by GALE, is involved in galactose metabolism and protein glycosylation. Here, we studied 3 patients from 2 unrelated families who showed lifelong severe thrombocytopenia, bleeding diathesis, mental retardation, mitral valve prolapse, and jaundice. Whole-exome sequencing revealed 4 variants that affect GALE, 3 of those previously unreported (Pedigree A, p.Lys78ValfsX32 and p.Thr150Met; Pedigree B, p.Val128Met; and p.Leu223Pro). Platelet phenotype analysis showed giant and/or grey platelets, impaired platelet aggregation, and severely reduced alpha and dense granule secretion. Enzymatic activity of the UDP-galactose-4-epimerase enzyme was severely decreased in all patients. Immunoblotting of platelet lysates revealed reduced GALE protein levels, a significant decrease in N-acetyl-lactosamine (LacNAc), showing a hypoglycosylation pattern, reduced surface expression of gylcoprotein Ibα-IX-V (GPIbα-IX-V) complex and mature β1 integrin, and increased apoptosis. In vitro studies performed with patients-derived megakaryocytes showed normal ploidy and maturation but decreased proplatelet formation because of the impaired glycosylation of the GPIbα and β1 integrin, and reduced externalization to megakaryocyte and platelet membranes. Altered distribution of filamin A and actin and delocalization of the von Willebrand factor were also shown. Overall, this study expands our knowledge of GALE-related thrombocytopenia and emphasizes the critical role of GALE in the physiological glycosylation of key proteins involved in platelet production and function., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

MLL3, also known as KMT2C, is a lysine mono-methyltransferase in charge of the writing of an epigenetic mark on lysine 4 from histone 3. The catalytic site of MLL3 is composed of four tyrosines, namely, Y44, Y69, Y128, and Y130. Tyrosine residues are highly conserved among lysine methyltransferases' catalytic sites, although their complete function is still unclear. The exploration of how modifications on these residues from the enzymatic machinery impact the enzymatic activity of MLL3 could shed light transversally into the inner functioning of enzymes with similar characteristics. Through the use of QMMM calculations, we focus on the effect of the mutation of each tyrosine from the catalytic site on the enzymatic activity and the product specificity in the current study. While we found that the mutations of Y44 and Y128 by phenylalanine inactivated the enzyme, the mutation of Y128 by alanine reactivated the enzymatic activity of MLL3. Moreover, according to our models, the Y128A mutant was even found to be capable of di- and tri-methylate lysine 4 from histone 3, what would represent a gain of function mutation, and could be responsible for the development of diseases. Finally, we were able to establish the inactivation mechanism, which involved the use of Y130 as a water occlusion structure, whose conformation, once perturbed by its mutation or Y128 mutant, allows the access of water molecules that sequester the electron pair from lysine 4 avoiding its methylation process and, thus, increasing the barrier height.

DNA damage is ubiquitous in nature and is at the basis of emergent treatments such as photodynamic therapy, which is based on the activation of highly oxidative reactive oxygen species by photosensitizing O 2 . However, hypoxia observed in solid tumors imposes the necessity to devise oxygen-independent modes of action able to induce DNA damage under a low oxygen concentration. The complexity of these DNA damage mechanisms in realistic environments grows exponentially when taking into account light absorption and subsequent excited-state population, photochemical and (photo)-redox reactions, the multiple species involved in different electronic states, noncovalent interactions, multiple reaction steps, and the large number of DNA reactive sites. This work tackles all the intricate reactivity of a photosensitizer based on a nitroimidazole derivative reacting toward DNA in solution under UV light exposition. This is performed through a combination of ground- and excited-state quantum chemistry, classical molecular dynamics, and hybrid QM/MM simulations to rationalize in detail the formation of DNA interstrand cross-links (ICLs) exerted by the noncanonical noncovalent photosensitizer. Unprecedented spatial and temporal resolution of these phenomena is achieved, revealing that the ICL is sequence-specific and that the fastest reactions take place at AT, GC, and GT steps involving either the opposite nucleobases or adjacent Watson-Crick base pairs. The N7 and O6 positions of guanine, the N7 and N3 sites of adenine, the N4 position of cytosine, and the O2 atom of thymine are deemed as the most nucleophile sites and are positively identified to participate in the ICL productions. This work provides a multiscale computational protocol to study DNA reactivity with noncovalent photosensitizers, and contributes to the understanding of therapies based on photoinduced DNA damage at molecular and electronic levels. In addition, we believe the depth understanding of these processes should assist the design of new photosensitizers considering their molecular size, electronic properties, and the observed regioselectivity toward nucleic acids.

Cysteine proteases are an important target for the development of inhibitors that could be used as drugs to regulate the activity of these kinds of enzymes involved in many diseases, including COVID-19. For this reason, it is important to have methodological tools that allow a detailed study of their activity and inhibition, combining computational efficiency and accuracy. We here explore the performance of different quantum mechanics/molecular mechanics methods to explore the inhibition reaction mechanism of the SARS-CoV-2 3CL protease with a hydroxymethyl ketone derivative. We selected two density functional theory (DFT) functionals (B3LYP and M06-2X), two semiempirical Hamiltonians (AM1d and PM6), and two tight-binding DFT methods (DFTB3 and GFN2-xTB) to explore the free energy landscape associated with this reaction. We show that it is possible to obtain an accurate description combining molecular dynamics simulations performed using tight-binding DFT methods and single-point energy corrections at a higher QM description. The use of a computational strategy that provides reliable results at a reasonable computational cost could assist the in silico screening of possible candidates during the design of new drugs directed against cysteine proteases.

Chronic myeloid leukaemia (CML) is a haematological neoplasm driven by the BCR/ABL fusion oncogene. The monogenic aspect of the disease and the feasibility of ex vivo therapies in haematological disorders make CML an excellent candidate for gene therapy strategies. The ability to abolish any coding sequence by CRISPR-Cas9 nucleases offers a powerful therapeutic opportunity to CML patients. However, a definitive cure can only be achieved when only CRISPR-edited cells are selected. A gene-trapping approach combined with CRISPR technology would be an ideal approach to ensure this. Here, we developed a CRISPR-Trap strategy that efficiently inserts a donor gene trap (SA-CMV-Venus) cassette into the BCR/ABL -specific fusion point in the CML K562 human cell line. The trapping cassette interrupts the oncogene coding sequence and expresses a reporter gene that enables the selection of edited cells. Quantitative mRNA expression analyses showed significantly higher level of expression of the BCR/Venus allele coupled with a drastically lower level of BCR/ABL expression in Venus + cell fractions. Functional in vitro experiments showed cell proliferation arrest and apoptosis in selected Venus + cells. Finally, xenograft experiments with the selected Venus + cells showed a large reduction in tumour growth, thereby demonstrating a therapeutic benefit in vivo. This study represents proof of concept for the therapeutic potential of a CRISPR-Trap system as a novel strategy for gene elimination in haematological neoplasms.

Background: Rare inherited thrombocytopenias are caused by alterations in genes involved in megakaryopoiesis, thrombopoiesis and/or platelet release. Diagnosis is challenging due to poor specificity of platelet laboratory assays, large numbers of culprit genes, and difficult assessment of the pathogenicity of novel variants., Objectives: To characterize the clinical and laboratory phenotype, and identifying the underlying molecular alteration, in a pedigree with thrombocytopenia of uncertain etiology., Patients/methods: Index case was enrolled in our Spanish multicentric project of inherited platelet disorders due to lifelong thrombocytopenia and bleeding. Bleeding score was recorded by ISTH-BAT. Laboratory phenotyping consisted of blood cells count, blood film, platelet aggregation and flow cytometric analysis. Genotyping was made by whole-exome sequencing (WES). Cytoskeleton proteins were analyzed in resting/spreading platelets by immunofluorescence and immunoblotting., Results: Five family members displayed lifelong mild thrombocytopenia with a high number of enlarged platelets in blood film, and mild bleeding tendency. Patient's platelets showed normal aggregation and granule secretion response to several agonists. WES revealed a novel nonsense variant (c.322C>T; p.Gln108*) in TPM4 (NM_003290.3), the gene encoding for tropomyosin-4 (TPM4). This variant led to impairment of platelet spreading capacity after stimulation with TRAP-6 and CRP, delocalization of TPM4 in activated platelets, and significantly reduced TPM4 levels in platelet lysates. Moreover, the index case displayed up-regulation of TPM2 and TPM3 mRNA levels., Conclusions: This study identifies a novel TPM4 nonsense variant segregating with macrothrombocytopenia and impaired platelet cytoskeletal remodeling and spreading. These findings support the relevant role of TPM4 in thrombopoiesis and further expand our knowledge of TPM4-related thrombocytopenia., (© 2022 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis.)

We present the results of classical and QM/MM simulations for the inhibition of SARS-CoV-2 3CL protease by a hydroxymethylketone inhibitor, PF-00835231. In the noncovalent complex the carbonyl oxygen atom of the warhead is placed in the oxyanion hole formed by residues 143 to 145, while P1-P3 groups are accommodated in the active site with interactions similar to those observed for the peptide substrate. According to alchemical free energy calculations, the P1' hydroxymethyl group also contributes to the binding free energy. Covalent inhibition of the enzyme is triggered by the proton transfer from Cys145 to His41. This step is followed by the nucleophilic attack of the Sγ atom on the carbonyl carbon atom of the inhibitor and a proton transfer from His41 to the carbonyl oxygen atom mediated by the P1' hydroxyl group. Computational simulations show that the addition of a chloromethyl substituent to the P1' group may lower the activation free energy for covalent inhibition., (© 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Competing Interests: The authors declare no conflict of interest.

We present a detailed computational analysis of the binding mode and reactivity of the novel oral inhibitor PF-07321332 developed against the SARS-CoV-2 3CL protease. Alchemical free energy calculations suggest that positions P3 and P4 could be susceptible to improvement in order to get a larger binding strength. QM/MM simulations unveil the reaction mechanism for covalent inhibition, showing that the nitrile warhead facilitates the recruitment of a water molecule for the proton transfer step.

RUNX1 -related disorder ( RUNX1 -RD) is caused by germline variants affecting the RUNX1 gene. This rare, heterogeneous disorder has no specific clinical or laboratory phenotype, making genetic diagnosis necessary. Although international recommendations have been established to classify the pathogenicity of variants, identifying the causative alteration remains a challenge in RUNX1 -RD. Murine models may be useful not only for definitively settling the controversy about the pathogenicity of certain RUNX1 variants, but also for elucidating the mechanisms of molecular pathogenesis. Therefore, we developed a knock-in murine model, using the CRISPR/Cas9 system, carrying the RUNX1 p.Leu43Ser variant (mimicking human p.Leu56Ser) to study its pathogenic potential and mechanisms of platelet dysfunction. A total number of 75 mice were generated; 25 per genotype (RUNX1 WT/WT , RUNX1 WT/L43S , and RUNX1 L43S/L43S ). Platelet phenotype was assessed by flow cytometry and confocal microscopy. On average, RUNX1 L43S/L43S and RUNX1 WT/L43S mice had a significantly longer tail-bleeding time than RUNX1 WT/WT mice, indicating the variant's involvement in hemostasis. However, only homozygous mice displayed mild thrombocytopenia. RUNX1 L43S/L43S and RUNX1 WT/L43S displayed impaired agonist-induced spreading and α-granule release, with no differences in δ-granule secretion. Levels of integrin α IIb β 3 activation, fibrinogen binding, and aggregation were significantly lower in platelets from RUNX1 L43S/L43S and RUNX1 WT/L43S using phorbol 12-myristate 13-acetate (PMA), adenosine diphosphate (ADP), and high thrombin doses. Lower levels of PKC phosphorylation in RUNX1 L43S/L43S and RUNX1 WT/L43S suggested that the PKC-signaling pathway was impaired. Overall, we demonstrated the deleterious effect of the RUNX1 p.Leu56Ser variant in mice via the impairment of integrin α IIb β 3 activation, aggregation, α-granule secretion, and platelet spreading, mimicking the phenotype associated with RUNX1 variants in the clinical setting., Competing Interests: None declared., (Thieme. All rights reserved.)

Haematopoiesis is a paradigm of cell differentiation because of the wide variety and overwhelming number of mature blood cells produced daily. Under stress conditions, the organism must adapt to a boosted demand for blood cells. Chronic granulomatous disease (CGD) is a genetic disease caused by inactivating mutations that affect the phagocyte oxidase. Besides a defective innate immune system, CGD patients suffer from recurrent hyper-inflammation episodes, circumstances upon which they must face emergency haematopoiesis. The targeting of Cybb and Ncf1 genes have produced CGD animal models that are a useful surrogate when studying the pathophysiology and treatment of this disease. Here, we show that Cyba -/- mice spontaneously develop granuloma and, therefore, constitute a CGD animal model to complement the existing Cybb -/- and Ncf1 -/- models. More importantly, we have analysed haematopoiesis in granuloma-bearing Cyba -/- mice. These animals showed a significant loss of weight, developed remarkable splenomegaly, bone marrow myeloid hyperplasia, and signs of anaemia. Haematological analyses showed a sharped decrease of B-cells and a striking development of myeloid cells in all compartments. Collectively, our results show that granuloma inflammatory lesions dramatically change haematopoiesis homeostasis. Consequently, we suggest that besides their defective innate immunity, the alteration of haematopoiesis homeostasis upon granuloma may contribute to the dismal outcome of CGD.

Chronic myeloid leukemia (CML) is a hematopoietic malignancy produced by a unique oncogenic event involving the constitutively active tyrosine-kinase (TK) BCR/ABL1 . TK inhibitors (TKI) changed its prognosis and natural history. Unfortunately, ABL1 remains unaffected by TKIs. Leukemic stem cells (LSCs) remain, and resistant mutations arise during treatment. To address this problem, we have designed a therapeutic CRISPR-Cas9 deletion system targeting BCR/ABL1 . The system was efficiently electroporated to cell lines, LSCs from a CML murine model, and LSCs from CML patients at diagnosis, generating a specific ABL1 null mutation at high efficiency and allowing the edited leukemic cells to be detected and tracked. The CRISPR-Cas9 deletion system triggered cell proliferation arrest and apoptosis in murine and human CML cell lines. Patient and murine-derived xenografts with CRISPR-edited LSCs in NOD SCID gamma niches revealed that normal multipotency and repopulation ability of CRISPR edited LSCs were fully restored. Normal hematopoiesis was restored, avoiding myeloid bias. To the best of our knowledge, we show for the first time how a CRISPR-Cas9 deletion system efficiently interrupts BCR/ABL1 oncogene in primary LSCs to bestow a therapeutic benefit. This study is a proof of concept for genome editing in all those diseases, like CML, sustained by a single oncogenic event, opening up new therapeutic opportunities.

The mixed lineage leukemia 3 or MLL3 is the enzyme in charge of the writing of an epigenetic mark through the methylation of lysine 4 from the N-terminal domain of histone 3 and its deregulation has been related to several cancer lines. An interesting feature of this enzyme comes from its regulation mechanism, which involves its binding to an activating dimer before it can be catalytically functional. Once the trimer is formed, the reaction mechanism proceeds through the deprotonation of the lysine followed by the methyl-transfer reaction. Here we present a detailed exploration of the activation mechanism through a QM/MM approach focusing on both steps of the reaction, aiming to provide new insights into the deprotonation process and the role of the catalytic machinery in the methyl-transfer reaction. Our finding suggests that the source of the activation mechanism comes from conformational restriction mediated by the formation of a network of salt-bridges between MLL3 and one of the activating subunits, which restricts and stabilizes the positioning of several residues relevant for the catalysis. New insights into the deprotonation mechanism of lysine are provided, identifying a valine residue as crucial in the positioning of the water molecule in charge of the process. Finally, a tyrosine residue was found to assist the methyl transfer from SAM to the target lysine.

BIRC3 is monoallelically deleted in up to 80% of chronic lymphocytic leukemia (CLL) cases harboring del(11q). In addition, truncating mutations in the remaining allele of this gene can lead to BIRC3 biallelic inactivation, which has been shown to be a marker for reduced survival in CLL. Nevertheless, the biological mechanisms by which these lesions could contribute to del(11q) CLL pathogenesis and progression are partially unexplored. We implemented the CRISPR/Cas9-editing system to generate isogenic CLL cell lines harboring del(11q) and/or BIRC3 mutations, modeling monoallelic and biallelic BIRC3 loss. Our results reveal that monoallelic BIRC3 deletion in del(11q) cells promotes non-canonical NF-κB signaling activation via RelB-p52 nuclear translocation, being these effects allelic dose-dependent and therefore further enhanced in del(11q) cells with biallelic BIRC3 loss. Moreover, we demonstrate ex vivo in primary cells that del(11q) cases including BIRC3 within their deleted region show evidence of non-canonical NF-κB activation which correlates with high BCL2 levels and enhanced sensitivity to venetoclax. Furthermore, our results show that BIRC3 mutations in del(11q) cells promote clonal advantage in vitro and accelerate leukemic progression in an in vivo xenograft model. Altogether, this work highlights the biological bases underlying disease progression of del(11q) CLL patients harboring BIRC3 deletion and mutation., (© 2021. The Author(s).)

Janus kinases (JAKs) are a family of proinflammatory enzymes able to mediate the immune responses and the inflammatory cascade by modulating multiple cytokine expressions as well as various growth factors. In the present study, the inhibition of the JAK-signal transducer and activator of transcription (STAT) signaling pathway is explored as a potential strategy for treating autoimmune and inflammatory disorders. A computationally driven approach aimed at identifying novel JAK inhibitors based on molecular topology, docking, and molecular dynamics simulations was carried out. For the best candidates selected, the inhibitory activity against JAK2 was evaluated in vitro . Two hit compounds with a novel chemical scaffold, 4 (IC 50 = 0.81 μM) and 7 (IC 50 = 0.64 μM), showed promising results when compared with the reference drug Tofacitinib (IC 50 = 0.031 μM).

Limbal stem cells (LSC) maintain the transparency of the corneal epithelium. Chemical burns lead the loss of LSC inducing an up-regulation of pro-inflammatory and pro-angiogenic factors, triggering corneal neovascularization and blindness. Adipose tissue-derived mesenchymal stem cells (AT-MSC) have shown promise in animal models to treat LSC deficiency (LSCD), but there are not studies showing their efficacy when primed with different media before transplantation. We cultured AT-MSC with standard medium and media used to culture LSC for clinical application. We demonstrated that different media changed the AT-MSC paracrine secretion showing different paracrine effector functions in an in vivo model of chemical burn and in response to a novel in vitro model of corneal inflammation by alkali induction. Treatment of LSCD with AT-MSC changed the angiogenic and inflammatory cytokine profile of mice corneas. AT-MSC cultured with the medium that improved their cytokine secretion, enhanced the anti-angiogenic and anti-inflammatory profile of the treated corneas. Those corneas also presented better outcome in terms of corneal transparency, neovascularization and histologic reconstruction. Priming human AT-MSC with LSC specific medium can potentiate their ability to improve corneal wound healing, decrease neovascularization and inflammation modulating paracrine effector functions in an in vivo optimized rat model of LSCD., (© 2021 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

We here investigate the mechanism of SARS-CoV-2 3CL protease inhibition by one of the most promising families of inhibitors, those containing an aldehyde group as a warhead. These compounds are covalent inhibitors that inactivate the protease, forming a stable hemithioacetal complex. Inhibitor 11a is a potent inhibitor that has been already tested in vitro and in animals. Using a combination of classical and QM/MM simulations, we determined the binding mode of the inhibitor into the active site and the preferred rotameric state of the catalytic histidine. In the noncovalent complex, the aldehyde group is accommodated into the oxyanion hole formed by the NH main-chain groups of residues 143 to 145. In this pose, P1-P3 groups of the inhibitor mimic the interactions established by the natural peptide substrate. The reaction is initiated with the formation of the catalytic dyad ion pair after a proton transfer from Cys145 to His41. From this activated state, covalent inhibition proceeds with the nucleophilic attack of the deprotonated Sγ atom of Cys145 to the aldehyde carbon atom and a water-mediated proton transfer from the Nε atom of His41 to the aldehyde oxygen atom. Our proposed reaction transition-state structure is validated by comparison with X-ray data of recently reported inhibitors, while the activation free energy obtained from our simulations agrees with the experimentally derived value, supporting the validity of our findings. Our study stresses the interplay between the conformational dynamics of the inhibitor and the protein with the inhibition mechanism and the importance of including conformational diversity for accurate predictions about the inhibition of the main protease of SARS-CoV-2. The conclusions derived from our work can also be used to rationalize the behavior of other recently proposed inhibitor compounds, including aldehydes and ketones with high inhibitory potency., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.)

The constitutively active tyrosine-kinase BCR/ABL1 oncogene plays a key role in human chronic myeloid leukemia development and disease maintenance, and determines most of the features of this leukemia. For this reason, tyrosine-kinase inhibitors are the first-line treatment, offering most patients a life expectancy like that of an equivalent healthy person. However, since the oncogene stays intact, lifelong oral medication is essential, even though this triggers adverse effects in many patients. Furthermore, leukemic stem cells remain quiescent and resistance is observed in approximately 25% of patients. Thus, new therapeutic alternatives are still needed. In this scenario, the interruption/deletion of the oncogenic sequence might be an effective therapeutic option. The emergence of CRISPR (clustered regularly interspaced short palindromic repeats) technology can offer a definitive treatment based on its capacity to induce a specific DNA double strand break. Besides, it has the advantage of providing complete and permanent oncogene knockout, while tyrosine kinase inhibitors (TKIs) only ensure that BCR-ABL1 oncoprotein is inactivated during treatment. CRISPR/Cas9 cuts DNA in a sequence-specific manner making it possible to turn oncogenes off in a way that was not previously feasible in humans. This review describes chronic myeloid leukemia (CML) disease and the main advances in the genome-editing field by which it may be treated in the future.

Communications, navigation, and other related systems need to have a well-defined antenna radiation pattern. In onboard vessel systems, the radiation pattern can be much different than the one obtained for an isolated antenna (because of the vessel's structure and other nearby radiating systems interference). Finding out the onboard antenna's radiation pattern is a well-known problem for any shipbuilder/owner. The conventional method consists of measuring radiation patterns from a fixed antenna on the coast while the ship is navigating in circles. Recent electronic systems in the market now allow for an alternative method: keeping the ship static while an unmanned aerial vehicle (UAV) circles it, measuring the antenna's transmitted power. This research paper examines the airspace volume and optimal flight path of an off-the-shelf UAV system for measuring the onboard antenna's radiation pattern in the presence of physical constraints such as the vessel's dimensions, safety zone, distance to base, Fresnel's and multipath distances, and considering the loss due to polarization decoupling between the antenna under test and UAV's antenna., Competing Interests: NO authors have competing interests.

The irreversible inhibition of the main protease of SARS-CoV-2 by a Michael acceptor known as N3 has been investigated using multiscale methods. The noncovalent enzyme-inhibitor complex was simulated using classical molecular dynamics techniques and the pose of the inhibitor in the active site was compared to that of the natural substrate, a peptide containing the Gln-Ser scissile bond. The formation of the covalent enzyme-inhibitor complex was then simulated using hybrid QM/MM free energy methods. After binding, the reaction mechanism was found to be composed of two steps: (i) the activation of the catalytic dyad (Cys145 and His41) to form an ion pair and (ii) a Michael addition where the attack of the Sγ atom of Cys145 to the Cβ atom of the inhibitor precedes the water-mediated proton transfer from His41 to the Cα atom. The microscopic description of protease inhibition by N3 obtained from our simulations is strongly supported by the excellent agreement between the estimated activation free energy and the value derived from kinetic experiments. Comparison with the acylation reaction of a peptide substrate suggests that N3-based inhibitors could be improved by adding chemical modifications that could facilitate the formation of the catalytic dyad ion pair., Competing Interests: The are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

The regulation of protein function by reversible oxidation is increasingly recognized as a key mechanism for the control of cellular signaling, modulating crucial biological processes such as cell differentiation. In this scenario, NADPH oxidases must occupy a prominent position. Our results show that hematopoietic stem and progenitor cells express three p22phox-dependent NADPH oxidases members (NOX1, NOX2 and NOX4). By deleting the p22phox coding gene (Cyba), here we have analyzed the importance of this family of enzymes during in vivo hematopoiesis. Cyba-/- mice show a myeloid bias, and an enrichment of hematopoietic stem cell populations. By means of hematopoietic transplant experiments we have also tried to dissect the specific role of the NADPH oxidases. While the absence of NOX1 or NOX2 provides a higher level of reconstitution, a lack of NOX4 rendered the opposite result, suggesting a functional specificity among the different NADPH oxidases. Cyba-/- cells showed a hampered activation of AKT1 and a sharp decrease in STAT5 protein. This is in line with the diminished response to IL-7 shown by our results, which could explain the overproduction of immunoglobulins observed in Cyba-/- mice.

Herein we present the results of an in-depth simulation study of LinA and its two variants. In our analysis, we combined the exploration of protein conformational dynamics with and without bound substrates (hexachlorocyclohexane (HCH) isomers) performed using molecular dynamics simulation followed by the extraction of the most frequently visited conformations and their characteristics with a detailed description of the interactions taking place in the active site between the respective HCH molecule and the first shell residues by using symmetry-adapted perturbation theory (SAPT) calculations. A detailed investigation of the conformational space of LinA substates has been accompanied by description of enzymatic catalytic steps carried out using a hybrid quantum mechanics/molecular mechanics (QM/MM) potential along with the computation of the potential of mean force (PMF) to estimate the free energy barriers for the studied transformations: dehydrochlorination of γ-, (-)-α-, and (+)-α-HCH by LinA-type I and -type II variants. The applied combination of computational techniques allowed us not only to characterize two LinA types but also to point to the most important differences between them and link their features to catalytic efficiency each of them possesses toward the respective ligand. More importantly it has been demonstrated that type I protein is more mobile, its active site has a larger volume, and the dehydrochlorination products are stabilized more strongly than in the case of type II enzyme, due to differences in the residues present in the active sites. Additionally, interaction energy calculations revealed very interesting patterns not predicted before but having the potential to be utilized in any attempts of improving LinA catalytic efficiency. On the basis of all these observations, LinA-type I protein seems to be more preorganized for the dehydrochlorination reaction it catalyzes than the type II variant.

The deletion of 11q (del(11q)) invariably comprises ATM gene in chronic lymphocytic leukemia (CLL). Concomitant mutations in this gene in the remaining allele have been identified in 1/3 of CLL cases harboring del(11q), being the biallelic loss of ATM associated with adverse prognosis. Although the introduction of targeted BCR inhibition has significantly favored the outcomes of del(11q) patients, responses of patients harboring ATM functional loss through biallelic inactivation are unexplored, and the development of resistances to targeted therapies have been increasingly reported, urging the need to explore novel therapeutic approaches. Here, we generated isogenic CLL cell lines harboring del(11q) and ATM mutations through CRISPR/Cas9-based gene-editing. With these models, we uncovered a novel therapeutic vulnerability of del(11q)/ATM-mutated cells to dual BCR and PARP inhibition. Ex vivo studies in the presence of stromal stimulation on 38 CLL primary samples confirmed a synergistic action of the combination of olaparib and ibrutinib in del(11q)/ATM-mutated CLL patients. In addition, we showed that ibrutinib produced a homologous recombination repair impairment through RAD51 dysregulation, finding a synergistic link of both drugs in the DNA damage repair pathway. Our data provide a preclinical rationale for the use of this combination in CLL patients with this high-risk cytogenetic abnormality.

The Nomo1 gene mediates a wide range of biological processes of importance in embryonic development. Accordingly, constitutive perturbation of Nomo1 function may result in myriad developmental defects that trigger embryonic lethality. To extend our understanding of Nomo1 function in postnatal stages and in a tissue-specific manner, we generated a conditional knockout mouse model of Nomo1. To achieve this, we used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology in C57Bl/6J mouse zygotes to generate a new mouse model in which exon 3 of the Nomo1 gene is specifically flanked (or floxed) by LoxP sites (Nomo1 f/f ). Nomo1 f/f mouse embryonic fibroblasts were transduced with a Cre adenovirus and efficiently recombined between LoxP sites. Genomic and expression studies in Nomo1-transduced MEFs demonstrated that the Nomo1 exon 3 is ablated. Western blot assay showed that no protein or early truncated protein is produced. In vivo assay crossing Nomo1 f/f mouse with a Msi1-CRE transgenic mouse corroborated the previous findings and it showed Nomo1 exon 3 deletion at msi1+ cell compartment. This short technical report demonstrates that CRISPR/Cas9 technology is a simple and easy method for creating conditional mouse models. The Nomo1 f/f mouse will be useful to researchers who wish to explore the role of Nomo1 in any developmental stage or in a tissue-specific manner.