Population Dynamics and Structural Effects at Short and Long Range Support the Hypothesis of the Selective Advantage of the G614 SARS-CoV-2 Spike Variant.

SARS-CoV-2 epidemics quickly propagated worldwide, sorting virus genomic variants in newly established propagules of infections. Stochasticity in transmission within and between countries or an actual selective advantage could explain the global high frequency reached by some genomic variants. Using statistical analyses, demographic reconstructions, and molecular dynamics simulations, we show that the globally invasive G614 spike variant 1) underwent a significant demographic expansion in most countries explained neither by stochastic effects nor by overrepresentation in clinical samples, 2) increases the spike S1/S2 furin-like site conformational plasticity (short-range effect), and 3) modifies the internal motion of the receptor-binding domain affecting its cross-connection with other functional domains (long-range effect). Our results support the hypothesis of a selective advantage at the basis of the spread of the G614 variant, which we suggest may be due to structural modification of the spike protein at the S1/S2 proteolytic site, and provide structural information to guide the design of variant-specific drugs.

Genomic Signature of Shifts in Selection in a Subalpine Ant and Its Physiological Adaptations.

Understanding how organisms adapt to extreme environments is fundamental and can provide insightful case studies for both evolutionary biology and climate-change biology. Here, we take advantage of the vast diversity of lifestyles in ants to identify genomic signatures of adaptation to extreme habitats such as high altitude. We hypothesized two parallel patterns would occur in a genome adapting to an extreme habitat: 1) strong positive selection on genes related to adaptation and 2) a relaxation of previous purifying selection. We tested this hypothesis by sequencing the high-elevation specialist Tetramorium alpestre and four other phylogenetically related species. In support of our hypothesis, we recorded a strong shift of selective forces in T. alpestre, in particular a stronger magnitude of diversifying and relaxed selection when compared with all other ants. We further disentangled candidate molecular adaptations in both gene expression and protein-coding sequence that were identified by our genome-wide analyses. In particular, we demonstrate that T. alpestre has 1) a higher level of expression for stv and other heat-shock proteins in chill-shock tests and 2) enzymatic enhancement of Hex-T1, a rate-limiting regulatory enzyme that controls the entry of glucose into the glycolytic pathway. Together, our analyses highlight the adaptive molecular changes that support colonization of high-altitude environments.

Design of Disruptors of the Hsp90-Cdc37 Interface.

The molecular chaperone Hsp90 is a ubiquitous ATPase-directed protein responsible for the activation and structural stabilization of a large clientele of proteins. As such, Hsp90 has emerged as a suitable candidate for the treatment of a diverse set of diseases, such as cancer and neurodegeneration. The inhibition of the chaperone through ATP-competitive inhibitors, however, was shown to lead to undesirable side effects. One strategy to alleviate this problem is the development of molecules that are able to disrupt specific protein-protein interactions, thus modulating the activity of Hsp90 only in the particular cellular pathway that needs to be targeted. Here, we exploit novel computational and theoretical approaches to design a set of peptides that are able to bind Hsp90 and compete for its interaction with the co-chaperone Cdc37, which is found to be responsible for the promotion of cancer cell proliferation. In spite of their capability to disrupt the Hsp90-Cdc37 interaction, no important cytotoxicity was observed in human cancer cells exposed to designed compounds. These findings imply the need for further optimization of the compounds, which may lead to new ways of interfering with the Hsp90 mechanisms that are important for tumour growth.

The human DNA topoisomerase I mutant Gly717Asp: Higher religation rate is not always associated with camptothecin resistance.

DNA topoisomerases are key enzyme responsible for modulating the topological state of the DNA by breaking and rejoining of DNA strand. Characterization of a Gly717Asp mutation in the human topoisomerase was performed using several catalytic assays. The mutant enzyme was shown to have comparable cleavage and fast religation rate as compared to the wild-type protein. Addition of the anticancer drug camptothecin significantly reduced the religation step. The simulative approaches and analysis of the cleavage/religation equilibrium indicate that the mutation is able to modify the architecture of the drug binding site, increasing the persistence of the drug for the enzyme-DNA covalent complex. Taken together these results indicate that the structure modification of the drug binding site is the key reason for the increasing CPT persistence and furthermore provide the possibility for new anti-cancer drug discovery.

Computational Analysis of Dengue Virus Envelope Protein (E) Reveals an Epitope with Flavivirus Immunodiagnostic Potential in Peptide Microarrays.

The mosquito-borne viral disease caused by the Dengue virus is an expanding global threat. Diagnosis in low-resource-settings and epidemiological surveillance urgently requires new immunoprobes for serological tests. Structure-based epitope prediction is an efficient method to design diagnostic peptidic probes able to reveal specific antibodies elicited in response to infections in patients' sera. In this study, we focused on the Dengue viral envelope protein (E); computational analyses ranging from extensive Molecular Dynamics (MD) simulations and energy-decomposition-based prediction of potentially immunoreactive regions identified putative epitope sequences. Interestingly, one such epitope showed internal dynamic and energetic properties markedly different from those of other predicted sequences. The epitope was thus synthesized as a linear peptide, modified for chemoselective immobilization on microarrays and used in a serological assay to discriminate Dengue-infected individuals from healthy controls. The synthetic epitope probe showed a diagnostic performance comparable to that of the full antigen in terms of specificity and sensitivity. Given the high level of sequence identity among different flaviviruses, the epitope was immune-reactive towards Zika-infected sera as well. The results are discussed in the context of the quest for new possible structure-dynamics-based rules for the prediction of the immunoreactivity of selected antigenic regions with potential pan-flavivirus immunodiagnostic capacity.

Tyr120Asp mutation alters domain flexibility and dynamics of MeCP2 DNA binding domain leading to impaired DNA interaction: Atomistic characterization of a Rett syndrome causing mutation.

Mutations in the X-linked MECP2 gene represent the main origin of Rett syndrome, causing a profound intellectual disability in females. MeCP2 is an epigenetic transcriptional regulator containing two main functional domains: a methyl-CpG binding domain (MBD) and a transcription repression domain (TRD). Over 600 pathogenic mutations were reported to affect the whole protein; almost half of missense mutations affect the MBD. Understanding the impact of these mutations on the MBD structure and interaction with DNA will foster the comprehension of their pathogenicity and possibly genotype/phenotype correlation studies. Herein, we use molecular dynamics simulations to obtain a detailed view of the dynamics of WT and mutated MBD in the presence and absence of DNA. The pathogenic mutation Y120D is used as paradigm for our studies. Further, since the Y120 residue was previously found to be a phosphorylation site, we characterize the dynamic profile of the MBD also in the presence of Y120 phosphorylation (pY120). We found that addition of a phosphate group to Y120 or mutation in aspartic acid affect domain mobility that samples an alternative conformational space with respect to the WT, leading to impaired ability to interact with DNA. Experimental assays showing a significant reduction in the binding affinity between the mutated MBD and the DNA confirmed our predictions.

Chemo-enzymatic synthesis of (E)-2,3-diaryl-5-styryl-trans-2,3-dihydrobenzofuran-based scaffolds and their in vitro and in silico evaluation as a novel sub-family of potential allosteric modulators of the 90 kDa heat shock protein (Hsp90).

Herein we propose a facile, versatile and selective chemo-enzymatic synthesis of substituted (E)-2,3-diaryl-5-styryl-trans-2,3-dihydrobenzofurans based on the exploitation of the laccase-mediated oxidative (homo)coupling of (E)-4-styrylphenols. Thanks to this novel synthetic strategy, a library of benzofuran-based potential allosteric activators of the Heat shock protein 90 (Hsp90) was easily prepared. Moreover, considering their structural analogies to previously reported allosteric modulators, the sixteen new compounds synthesized in this work were tested in vitro for their potential stimulatory action on the ATPase activity of the molecular chaperone Hsp90. Combining experimental and computational results, we propose a mechanism of action for these compounds, and expand the structure-activity relationship (SAR) information available for benzofuran-based Hsp90 activators.

Design of Allosteric Stimulators of the Hsp90 ATPase as New Anticancer Leads.

Allosteric compounds that stimulate Hsp90 adenosine triphosphatase (ATPase) activity were rationally designed, showing anticancer potencies in the low micromolar to nanomolar range. In parallel, the mode of action of these compounds was clarified and a quantitative model that links the dynamic ligand-protein cross-talk to observed cellular and in vitro activities was developed. The results support the potential of using dynamics-based approaches to develop original mechanism-based cancer therapeutics.

Mutation of Gly717Phe in human topoisomerase 1B has an effect on enzymatic function, reactivity to the camptothecin anticancer drug and on the linker domain orientation.

Human topoisomerase 1B controls the topological state of supercoiled DNA allowing the progression of fundamental cellular processes. The enzyme, which is the unique molecular target of the natural anticancer compound camptothecin, acts by cleaving one DNA strand and forming a transient protein-DNA covalent adduct. In this work the role of the Gly717 residue, located in a α-helix structure bridging the active site and the linker domain, has been investigated mutating it in Phe. The mutation gives rise to drug resistance in vivo as observed through a viability assay of yeast cells. In vitro activity assays show that the mutant is characterized by a fast religation rate, only partially reduced by the presence of the drug. Comparative molecular dynamics simulations of the native and mutant proteins indicate that the mutation of Gly717 affects the motion orientation of the linker domain, changing its interaction with the DNA substrate, likely affecting the strand rotation and religation rate. The mutation also causes a slight rearrangement of the active site and of the drug binding site, providing an additional explanation for the lowered effect of camptothecin toward the mutant.

Human topoisomerase IB is a target of a thiosemicarbazone copper(II) complex.

The human topoisomerase IB inhibition and the antiproliferative activity of 3-(4-bromophenyl)-1-pyridin-2-ylprop-2-en-1-one thiosemicarbazone HPyCT4BrPh alone and its copper(II) complex [Cu(PyCT4BrPh)Cl] was investigated. [Cu(PyCT4BrPh)Cl] inhibits both the DNA cleavage and religation step of the enzyme, whilst the ligand alone does not display any effect. In addition we show that coordination to copper(II) improves the cytotoxicity of HPyCT4BrPh against THP-1 leukemia and MCF-7 breast cancer cells. The data indicate that the copper(II) thiosemicarbazone complex may hit human topoisomerase IB and that metal coordination can be useful to improve cytotoxicity of this versatile class of compounds.

Simulations of DNA topoisomerase 1B bound to supercoiled DNA reveal changes in the flexibility pattern of the enzyme and a secondary protein-DNA binding site.

Human topoisomerase 1B has been simulated covalently bound to a negatively supercoiled DNA minicircle, and its behavior compared to the enzyme bound to a simple linear DNA duplex. The presence of the more realistic supercoiled substrate facilitates the formation of larger number of protein-DNA interactions when compared to a simple linear duplex fragment. The number of protein-DNA hydrogen bonds doubles in proximity to the active site, affecting all of the residues in the catalytic pentad. The clamp over the DNA, characterized by the salt bridge between Lys369 and Glu497, undergoes reduced fluctuations when bound to the supercoiled minicircle. The linker domain of the enzyme, which is implicated in the controlled relaxation of superhelical stress, also displays an increased number of contacts with the minicircle compared to linear DNA. Finally, the more complex topology of the supercoiled DNA minicircle gives rise to a secondary DNA binding site involving four residues located on subdomain III. The simulation trajectories reveal significant changes in the interactions between the enzyme and the DNA for the more complex DNA topology, which are consistent with the experimental observation that the protein has a preference for binding to supercoiled DNA.

Characterization of the differences in the cyclopiazonic acid binding mode to mammalian and P. Falciparum Ca2+ pumps: a computational study.

Despite the investments in malaria research, an effective vaccine has not yet been developed and the causative parasites are becoming increasingly resistant to most of the available drugs. PfATP6, the sarco/endoplasmic reticulum Ca2+ pump (SERCA) of P. falciparum, has been recently genetically validated as a potential antimalarial target and cyclopiazonic acid (CPA) has been found to be a potent inhibitor of SERCAs in several organisms, including P. falciparum. In position 263, PfATP6 displays a leucine residue, whilst the corresponding position in the mammalian SERCA is occupied by a glutamic acid. The PfATP6 L263E mutation has been studied in relation to the artemisinin inhibitory effect on P. falciparum and recent studies have provided evidence that the parasite with this mutation is more susceptible to CPA. Here, we characterized, for the first time, the interaction of CPA with PfATP6 and its mammalian counterpart to understand similarities and differences in the mode of binding of the inhibitor to the two Ca2+ pumps. We found that, even though CPA does not directly interact with the residue in position 263, the presence of a hydrophobic residue in this position in PfATP6 rather than a negatively charged one, as in the mammalian SERCA, entails a conformational arrangement of the binding pocket which, in turn, determines a relaxation of CPA leading to a different binding mode of the compound. Our findings highlight differences between the plasmodial and human SERCA CPA-binding pockets that may be exploited to design CPA derivatives more selective toward PfATP6.

The human topoisomerase 1B Arg634Ala mutation results in camptothecin resistance and loss of inter-domain motion correlation.

Human topoisomerase 1B, the unique target of the natural anticancer compound camptothecin, catalyzes the unwinding of supercoiled DNA by introducing transient single strand nicks and providing covalent protein-DNA adducts. The functional properties and the drug reactivity of the single Arg634Ala mutant have been investigated in comparison to the wild type enzyme. The mutant is characterized by an identical relaxation and cleavage rate but it displays resistance to camptothecin as indicated by a viability assay of the yeast cells transformed with the mutated protein. The mutant also displays a very fast religation rate that is only partially reduced by the presence of the drug, suggesting that this is the main reason for its resistance. A comparative analysis of the structural-dynamical properties of the native and mutant proteins by molecular dynamics simulation indicates that mutation of Arg634 brings to a loss of motion correlation between the different domains and in particular between the linker and the C-terminal domain, containing the catalytic tyrosine residue. These results indicate that the loss of motion correlation and the drug resistance are two strongly correlated events.

Geometrical constraints limiting the poly(ADP-ribose) conformation investigated by molecular dynamics simulation.

Poly(ADP-ribosylation) is a post-transductional modification that regulates protein's function. Most of the proteins subjected to this control mechanism belong to machineries involved in DNA damage repair, or DNA interacting proteins. Poly(ADP-ribose) polymers are long chains of even 100 monomer length that can be branched at several positions but, not withstanding its importance, nothing is known concerning its structure. To understand, which are the geometrical parameters that confer to the polymer the structural constraints that determine its interaction with the target proteins, we have performed molecular dynamics of three chains of different length, made by 5, 25, and 30 units, the last one being branched. Analysis of the simulations allowed us to identify the main intra- and inter-monomer dihedral angles that govern the structure of the polymer that however, does not reach a unique definite conformation.

Molecular mechanism of the camptothecin resistance of Glu710Gly topoisomerase IB mutant analyzed in vitro and in silico.

BACKGROUND: DNA topoisomerases are key enzymes that modulate the topological state of DNA through the breaking and rejoining of DNA strands. Human topoisomerase IB can be inhibited by several compounds that act through different mechanisms, including clinically used drugs, such as the derivatives of the natural compound camptothecin that reversibly bind the covalent topoisomerase-DNA complex, slowing down the religation of the cleaved DNA strand, thus inducing cell death. Three enzyme mutations, which confer resistance to irinotecan in an adenocarcinoma cell line, were recently identified but the molecular mechanism of resistance was unclear. METHODS: The three resistant mutants have been investigated in S. cerevisiae model system following their viability in presence of increasing amounts of camptothecin. A systematical analysis of the different catalytic steps has been made for one of these mutants (Glu710Gly) and has been correlated with its structural-dynamical properties studied by classical molecular dynamics simulation. RESULTS: The three mutants display a different degree of camptothecin resistance in a yeast cell viability assay. Characterization of the different steps of the catalytic cycle of the Glu710Gly mutant indicated that its resistance is related to a high religation rate that is hardly affected by the presence of the drug. Analysis of the dynamic properties through simulation indicate that the mutant displays a much lower degree of correlation in the motion between the different protein domains and that the linker almost completely loses its correlation with the C-terminal domain, containing the active site tyrosine. CONCLUSIONS: These results indicate that a fully functional linker is required to confer camptothecin sensitivity to topoisomerase I since the destabilization of its structural-dynamical properties is correlated to an increase of religation rate and drug resistance.

A derivative of the natural compound kakuol affects DNA relaxation of topoisomerase IB inhibiting the cleavage reaction.

Topoisomerases IB are anticancer and antimicrobial targets whose inhibition by several natural and synthetic compounds has been documented over the last three decades. Here we show that kakuol, a natural compound isolated from the rhizomes of Asarum sieboldii, and a derivative analogue are able to inhibit the DNA relaxation mediated by the human enzyme. The analogue is the most efficient one and the inhibitory effect is enhanced upon pre-incubation with the enzyme. Analysis of the different steps of the catalytic cycle indicates that the inhibition occurs at the cleavage level and does not prevent DNA binding. Molecular docking shows that the compound preferentially binds near the active site at the bottom of the catalytic residue Tyr723, providing an atomistic explanation for its inhibitory activity.

ATP independent type IB topoisomerase of Leishmania donovani is stimulated by ATP: an insight into the functional mechanism.

Most type IB topoisomerases do not require ATP and Mg(2+) for activity. However, as shown previously for vaccinia topoisomerase I, we demonstrate that ATP stimulates the relaxation activity of the unusual heterodimeric type IB topoisomerase from Leishmania donovani (LdTOP1L/S) in the absence of Mg(2+). The stimulation is independent of ATP hydrolysis but requires salt as a co-activator. ATP binds to LdTOP1L/S and increases its rate of strand rotation. Docking studies indicate that the amino acid residues His93, Tyr95, Arg188 and Arg190 of the large subunit may be involved in ATP binding. Site directed mutagenesis of these four residues individually to alanine and subsequent relaxation assays reveal that the R190A mutant topoisomerase is unable to exhibit ATP-mediated stimulation in the absence of Mg(2+). However, the ATP-independent relaxation activities of all the four mutant enzymes remain unaffected. Additionally, we provide evidence that ATP binds LdTOP1L/S and modulates the activity of the otherwise ATP-independent enzyme. This study establishes ATP as an activator of LdTOP1L/S in the absence of Mg(2+).

SARS-CoV-2 multi-variant rapid detector based on graphene transistor functionalized with an engineered dimeric ACE2 receptor.

Reliable point-of-care (POC) rapid tests are crucial to detect infection and contain the spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The emergence of several variants of concern (VOC) can reduce binding affinity to diagnostic antibodies, limiting the efficacy of the currently adopted tests, while showing unaltered or increased affinity for the host receptor, angiotensin converting enzyme 2 (ACE2). We present a graphene field-effect transistor (gFET) biosensor design, which exploits the Spike-ACE2 interaction, the crucial step for SARS-CoV-2 infection. Extensive computational analyses show that a chimeric ACE2-Fragment crystallizable (ACE2-Fc) construct mimics the native receptor dimeric conformation. ACE2-Fc functionalized gFET allows in vitro detection of the trimeric Spike protein, outperforming functionalization with a diagnostic antibody or with the soluble ACE2 portion, resulting in a sensitivity of 20 pg/mL. Our miniaturized POC biosensor successfully detects B.1.610 (pre-VOC), Alpha, Beta, Gamma, Delta, Omicron (i.e., BA.1, BA.2, BA.4, BA.5, BA.2.75 and BQ.1) variants in isolated viruses and patient's clinical nasopharyngeal swabs. The biosensor reached a Limit Of Detection (LOD) of 65 cps/mL in swab specimens of Omicron BA.5. Our approach paves the way for a new and reusable class of highly sensitive, rapid and variant-robust SARS-CoV-2 detection systems.

Interaction between natural compounds and human topoisomerase I.

Eukaryotic topoisomerase I (Top1) is a monomeric enzyme that catalyzes the relaxation of supercoiled DNA during important processes including DNA replication, transcription, recombination and chromosome condensation. Human Top1 I is of significant medical interest since it is the unique cellular target of camptothecin (CPT), a plant alkaloid that rapidly blocks both DNA and RNA synthesis. In this review, together with CPT, we point out the interaction between human Top1 and some natural compounds, such us terpenoids, flavonoids, stilbenes and fatty acids. The drugs can interact with the enzyme at different levels perturbing the binding, cleavage, rotation or religation processes. Here we focus on different assays that can be used to identify the catalytic step of the enzyme inhibited by different natural compounds.

Evidences of a natively unfolded state for the human topoisomerase IB N-terminal domain.

The N-terminal domain of human topoisomerase IB has been expressed, purified and characterized by spectroscopic techniques. CD spectra as a function of concentration and pH indicate that the domain does not possess any defined secondary structure. The protein is probably in a natively unfolded state since its denaturation curve is indicative of a non-cooperative transition. Evidence of a partially folded structure comes from the fluorescence spectrum of ANS, whose intensity increases in presence of the domain. Indication of a partial structural arrangement of the domain comes also from the endogenous fluorescence of tryptophans that is centred at 350 nm in the native and shifts to 354 nm in the fully denaturated protein. Interestingly despite the poor structural degree, as also confirmed by a predictive approach, the domain efficiently binds DNA, suggesting that the absence of a defined 3D structure has a functional meaning that permits the domain to be available for the interaction with different molecular partners.