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.

Three different mutations in the DNA topoisomerase 1B in Leishmania infantum contribute to resistance to antitumor drug topotecan.

BACKGROUND: The evolution of drug resistance is one of the biggest challenges in leishmaniasis and has prompted the need for new antileishmanial drugs. Repurposing of approved drugs is a faster and very attractive strategy that is gaining supporters worldwide. Different anticancer topoisomerase 1B (TOP1B) inhibitors have shown strong antileishmanial activity and promising selective indices, supporting the potential repurposing of these drugs. However, cancer cells and Leishmania share the ability to become rapidly resistant. The aim of this study was to complete a whole-genome exploration of the effects caused by exposure to topotecan in order to highlight the potential mechanisms deployed by Leishmania to favor its survival in the presence of a TOP1B inhibitor. METHODS: We used a combination of stepwise drug resistance selection, whole-genome sequencing, functional validation, and theoretical approaches to explore the propensity of and potential mechanisms deployed by three independent clones of L. infantum to resist the action of TOP1B inhibitor topotecan. RESULTS: We demonstrated that L. infantum is capable of becoming resistant to high concentrations of topotecan without impaired growth ability. No gene deletions or amplifications were identified from the next-generation sequencing data in any of the three resistant lines, ruling out the overexpression of efflux pumps as the preferred mechanism of topotecan resistance. We identified three different mutations in the large subunit of the leishmanial TOP1B (Top1BF187Y, Top1BG191A, and Top1BW232R). Overexpression of these mutated alleles in the wild-type background led to high levels of resistance to topotecan. Computational molecular dynamics simulations, in both covalent and non-covalent complexes, showed that these mutations have an effect on the arrangement of the catalytic pentad and on the interaction of these residues with surrounding amino acids and DNA. This altered architecture of the binding pocket results in decreased persistence of topotecan in the ternary complex. CONCLUSIONS: This work helps elucidate the previously unclear potential mechanisms of topotecan resistance in Leishmania by mutations in the large subunit of TOP1B and provides a valuable clue for the design of improved inhibitors to combat resistance in both leishmaniasis and cancer. Our data highlights the importance of including drug resistance evaluation in drug discovery cascades.

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.

Visualizing the Dynamics of a Protein Folding Machinery: The Mechanism of Asymmetric ATP Processing in Hsp90 and its Implications for Client Remodelling.

The Hsp90 chaperone system interacts with a wide spectrum of client proteins, forming variable and dynamic multiprotein complexes that involve the intervention of cochaperone partners. Recent results suggest that the role of Hsp90 complexes is to establish interactions that suppress unwanted client activities, allow clients to be protected from degradation and respond to biochemical signals. Cryo-electron microscopy (cryoEM) provided the first key molecular picture of Hsp90 in complex with a kinase, Cdk4, and a cochaperone, Cdc37. Here, we use a combination of molecular dynamics (MD) simulations and advanced comparative analysis methods to elucidate key aspects of the functional dynamics of the complex, with different nucleotides bound at the N-terminal Domain of Hsp90. The results reveal that nucleotide-dependent structural modulations reverberate in a striking asymmetry of the dynamics of Hsp90 and identify specific patterns of long-range coordination between the nucleotide binding site, the client binding pocket, the cochaperone and the client. Our model establishes a direct atomic-resolution cross-talk between the ATP-binding site, the client region that is to be remodeled and the surfaces of the Cdc37-cochaperone.

Bioinformatics and Biosimulations as Toolbox for Peptides and Peptidomimetics Design: Where Are We?

Peptides and peptidomimetics are strongly re-emerging as amenable candidates in the development of therapeutic strategies against a plethora of pathologies. In particular, these molecules are extremely suitable to treat diseases in which a major role is played by protein-protein interactions (PPIs). Unlike small organic compounds, peptides display both a high degree of specificity avoiding secondary off-targets effects and a relatively low degree of toxicity. Further advantages are provided by the possibility to easily conjugate peptides to functionalized nanoparticles, so improving their delivery and cellular uptake. In many cases, such molecules need to assume a specific three-dimensional conformation that resembles the bioactive one of the endogenous ligand. To this end, chemical modifications are introduced in the polypeptide chain to constrain it in a well-defined conformation, and to improve the drug-like properties. In this context, a successful strategy for peptide/peptidomimetics design and optimization is to combine different computational approaches ranging from structural bioinformatics to atomistic simulations. Here, we review the computational tools for peptide design, highlighting their main features and differences, and discuss selected protocols, among the large number of methods available, used to assess and improve the stability of the functional folding of the peptides. Finally, we introduce the simulation techniques employed to predict the binding affinity of the designed peptides for their targets.

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.

Ligand Binding, Unbinding, and Allosteric Effects: Deciphering Small-Molecule Modulation of HSP90.

The molecular chaperone HSP90 oversees the functional activation of a large number of client proteins. Because of its role in multiple pathways linked to cancer and neurodegeneration, drug discovery targeting HSP90 has been actively pursued. Yet, a number of inhibitors failed to meet expectations due to induced toxicity problems. In this context, allosteric perturbation has emerged as an alternative strategy for the pharmacological modulation of HSP90 functions. Specifically, novel allosteric stimulators showed the interesting capability of accelerating HSP90 closure dynamics and ATPase activities while inducing tumor cell death. Here, we gain atomistic insight into the mechanisms of allosteric ligand recognition and their consequences on the functional dynamics of HSP90, starting from the fully unbound state. We integrate advanced computational sampling methods based on FunnelMetadynamics, with the analysis of internal dynamics of the structural ensembles visited during the simulations. We observe several binding/unbinding events, and from these, we derive an accurate estimation of the absolute binding free energy. Importantly, we show that different binding poses induce different dynamics states. Our work for the first time explicitly correlates HSP90 responses to binding/unbinding of an allosteric ligand to the modulation of functionally oriented protein motions.

Peptides for Infectious Diseases: From Probe Design to Diagnostic Microarrays.

Peptides and peptidomimetics have attracted revived interest regarding their applications in chemical biology over the last few years. Their chemical versatility, synthetic accessibility and the ease of storage and management compared to full proteins have made peptides particularly interesting in diagnostic applications, where they proved to efficiently recapitulate the molecular recognition properties of larger protein antigens, and were proven to be able to capture antibodies circulating in the plasma and serum of patients previously exposed to bacterial or viral infections. Here, we describe the development, integration and application of strategies for computational prediction and design, advanced chemical synthesis, and diagnostic deployment in multiplexed assays of peptide-based materials which are able to bind antibodies of diagnostic as well as therapeutic interest. By presenting successful applications of such an integrated strategy, we argue that they will have an ever-increasing role in both basic and clinical realms of research, where important advances can be expected in the next few years.

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.

Handling FMRP and its molecular partners: Structural insights into Fragile X Syndrome.

Fragile X Mental Retardation Protein (FMRP) is a RNA-binding protein (RBP) known to control different steps of mRNA metabolism, even though its complete function is not fully understood yet. Lack or mutations of FMRP lead to Fragile X Syndrome (FXS), the most common form of inherited intellectual disability and a leading monogenic cause of autism spectrum disorder (ASD). It is well established that FMRP has a multi-domain architecture, a feature that allows this RBP to be engaged in a large interaction network with numerous proteins and mRNAs or non-coding RNAs. Insights into the three-dimensional (3D) structure of parts of its three domains (N-terminus, central domain and C-terminus) were obtained using Nuclear Magnetic Resonance and X-ray diffraction, but the complete 3D arrangement of each domain with respect to the others is still missing. Here, we review the structural features of FMRP and of the network of its protein and RNA interactions. Understanding these aspects is the first necessary step towards the design of novel compounds for new therapeutic interventions in FXS.

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.

Allosteric Modulators of HSP90 and HSP70: Dynamics Meets Function through Structure-Based Drug Design.

Molecular chaperones HSP90 and HSP70 are essential regulators of the folding and activation of a disparate ensemble of client proteins. They function through ATP hydrolysis and the assembly of multiprotein complexes with cochaperones and clients. While their therapeutic relevance is recognized, important details underlying the links between ATP-dependent conformational dynamics and clients/cochaperones recruitment remain elusive. Allosteric modulators represent fundamental tools to obtain molecular insights into functional regulation. By selective perturbation of different aspects of HSP90/HSP70 activities, allosteric drugs can tune rather than completely inhibit signaling cascades, providing information on the relationships between structure-dynamics and function. Herein, we review advances in the design of HSP90 and HSP70 allosteric modulators. We consider inhibitors and activators in different biochemical and disease models. We discuss these compounds as probes to decipher the complexity of the chaperone machinery and that at the same time represent starting leads for the development of drugs against cancer and neurodegeneration.

A Novel Mecp2Y120D Knock-in Model Displays Similar Behavioral Traits But Distinct Molecular Features Compared to the Mecp2-Null Mouse Implying Precision Medicine for the Treatment of Rett Syndrome.

MeCP2 is a fundamental protein associated with several neurological disorders, including Rett syndrome. It is considered a multifunctional factor with a prominent role in regulating chromatin structure; however, a full comprehension of the consequences of its deficiency is still lacking. Here, we characterize a novel mouse model of Mecp2 bearing the human mutation Y120D, which is localized in the methyl-binding domain. As most models of Mecp2, the Mecp2Y120D mouse develops a severe Rett-like phenotype. This mutation alters the interaction of the protein with chromatin, but surprisingly, it also impairs its association with corepressors independently on the involved interacting domains. These features, which become overt mainly in the mature brain, cause a more accessible and transcriptionally active chromatin structure; conversely, in the Mecp2-null brain, we find a less accessible and transcriptionally inactive chromatin. By demonstrating that different MECP2 mutations can produce concordant neurological phenotypes but discordant molecular features, we highlight the importance of considering personalized approaches for the treatment of Rett syndrome.

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.

Enhancing Antibody Serodiagnosis Using a Controlled Peptide Coimmobilization Strategy.

Antigen immunoreactivity is often determined by surface regions defined by the 3D juxtapositions of amino acids stretches that are not continuous in the linear sequence. As such, mimicking an antigen immunoreactivity by means of putative linear peptide epitopes for diagnostic purposes is not trivial. Here we present a straightforward and robust method to extend the reach of immune-diagnostic probes design by copresenting peptides belonging to the same antigenic surface. In this case study focused on a computationally predicted Zika virus NS1 protein putative antigenic region, we reached a diagnostic confidence by the oriented and spatially controlled coimmobilization of peptide sequences found adjacent within the protein fold, that cooperatively interacted to provide enhanced immunoreactivity with respect to single linear epitopes. Through our method, we were able to differentiate Zika infected individuals from healthy controls. Remarkably, our strategy fits well with the requirements to build high-throughput screening platforms of linear and mixed peptide libraries, and it could possibly facilitate the rapid identification of conformational immunoreactive regions.

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.

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.

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.

A unique binding mode of the eukaryotic translation initiation factor 4E for guiding the design of novel peptide inhibitors.

The interaction between the eukaryotic translation initiation factor 4E (eIF4E) and eIF4E binding proteins (4E-BP) is a promising template for the inhibition of eIF4E and the treatment of diseases such as cancer and a spectrum of autism disorders, including the Fragile X syndrome (FXS). Here, we report an atomically detailed model of the complex between eIF4E and a peptide fragment of a 4E-BP, the cytoplasmic Fragile X interacting protein (CYFIP1). This model was generated using computer simulations with enhanced sampling from an alchemical replica exchange approach and validated using long molecular dynamics simulations. 4E-BP proteins act as post-transcriptional regulators by binding to eIF4E and preventing mRNA translation. Dysregulation of eIF4E activity has been linked to cancer, FXS, and autism spectrum disorders. Therefore, the study of the mechanism of inhibition of eIF4E by 4E-BPs is key to the development of drug therapies targeting this regulatory pathways. The results obtained in this work indicate that CYFIP1 interacts with eIF4E by an unique mode not shared by other 4E-BP proteins and elucidate the mechanism by which CYFIP1 interacts with eIF4E despite having a sequence binding motif significantly different from most 4E-BPs. Our study suggests an alternative strategy for the design of eIF4E inhibitor peptides with superior potency and specificity than currently available.