Molecular choreography: Unveiling the dynamic landscape of type IIA DNA topoisomerases before T-segment passage through all-atom simulations.

Type IIA DNA topoisomerases are molecular nanomachines responsible for controlling topological states of DNA molecules. Here, we explore the dynamic landscape of yeast topoisomerase IIA during key stages of its catalytic cycle, focusing in particular on the events preceding the passage of the T-segment. To this end, we generated six configurations of fully catalytic yeast topo IIA, strategically inserted a T-segment into the N-gate in relevant configurations, and performed all-atom simulations. The essential motion of topo IIA protein dimer was characterized by rotational gyrating-like movement together with sliding motion within the DNA-gate. Both appear to be inherent properties of the enzyme and an inbuilt feature that allows passage of the T-segment through the cleaved G-segment. Coupled dynamics of the N-gate and DNA-gate residues may be particularly important for controlled and smooth passage of the T-segment and consequently the prevention of DNA double-strand breaks. QTK loop residue Lys367, which interacts with ATP and ADP molecules, is involved in regulating the size and stability of the N-gate. The unveiled features of the simulated configurations provide insights into the catalytic cycle of type IIA topoisomerases and elucidate the molecular choreography governing their ability to modulate the topological states of DNA topology.

Unveiling the interdomain dynamics of type II DNA topoisomerase through all-atom simulations: Implications for understanding its catalytic cycle.

Type IIA DNA topoisomerases are complex molecular nanomachines that manage topological states of the DNA molecule in the cell and play a crucial role in cellular processes such as cell division and transcription. They are also established targets of cancer chemotherapy. Starting from the available crystal structure of a fully catalytic topoisomerase IIA homodimer from Saccharomyces cerevisiae, we constructed three states of this molecular motor primarily changing the configurations of the DNA segment bound in the DNA gate and performed µs-long all-atom molecular simulations. A comprehensive analysis revealed a sliding motion within the DNA gate and a teamwork between the N-gate and DNA gate that may be associated with the necessary molecular events that allow passage of the T-segment of DNA. The observed movement of the ATPase dimer relative to the DNA domain was reflected in different interaction patterns between the K-loops of the transducer domain and the B-A-B form of the bound DNA. Based on the obtained results, we mapped simulated configurations to the structures in the proposed catalytic cycle through which type IIA topoisomerases exert their function and discussed the possible transition events. The results extend our understanding of the mechanism of action of type IIA topoisomerases and provide an atomistic interpretation of some of the observed features of these molecular motors.

Disrupting Cu trafficking as a potential therapy for cancer.

Copper ions play a crucial role in various cellular biological processes. However, these copper ions can also lead to toxicity when their concentration is not controlled by a sophisticated copper-trafficking system. Copper dys-homeostasis has been linked to a variety of diseases, including neurodegeneration and cancer. Therefore, manipulating Cu-trafficking to trigger selective cancer cell death may be a viable strategy with therapeutic benefit. By exploiting combined in silico and experimental strategies, we identified small peptides able to bind Atox1 and metal-binding domains 3-4 of ATP7B proteins. We found that these peptides reduced the proliferation of cancer cells owing to increased cellular copper ions concentration. These outcomes support the idea of harming copper trafficking as an opportunity for devising novel anti-cancer therapies.

Balanced dual acting compounds targeting aromatase and estrogen receptor α as an emerging therapeutic opportunity to counteract estrogen responsive breast cancer.

Breast Cancer (BC) is a leading cause of death in women, currently affecting 13% of female population worldwide. First-line clinical treatments against Estrogen Receptor positive (ER+) BC rely on suppressing estrogen production, by inhibiting the aromatase (AR) enzyme, or on blocking estrogen-dependent pro-oncogenic signaling, by targeting Estrogen Receptor (ER) α with selective Modulators/Degraders (SERMs/SERDs). The development of dual acting molecules targeting AR and ERα represents a tantalizing alternative strategy to fight ER + BC, reducing the incidence of adverse effects and resistance onset that limit the effectiveness of these gold-standard therapies. Here, in silico design, synthesis, biological evaluation and an atomic-level characterization of the binding and inhibition mechanism of twelve structurally related drug-candidates enable the discovery of multiple compounds active on both AR and ERα in the sub-µM range. The best drug-candidate 3a displayed a balanced low-nanomolar IC50 towards the two targets, SERM activity and moderate selectivity towards a BC cell line. Moreover, most of the studied compounds reduced ERα levels, suggesting a potential SERD activity. This study dissects the key structural traits needed to obtain optimal dual acting drug-candidates, showing that multitarget compounds may be a viable therapeutic option to counteract ER + BC.

A Rapid Prototyped Thermal Mass Flowmeter.

An innovative rapid prototyping technique for embedding microcomponents in PDMS replicas was developed and applied on a thermal mass flowmeter for closed loop micropump flowrate control. Crucial flowmeter design and thermal parameters were investigated with a 3-D fully coupled electro-thermal-fluidic model which was built in Comsol Multiphysics 5.2. The flowmeter was characterized for three distinct measuring configurations. For precise low flowrate applications, a sensor-heater-sensor flowmeter configuration with a constant heater temperature was found to be the most appropriate yielding the measuring range of 0 to 90 µL·min-1 and the sensitivity of 1.3 °C·µL-1·min in the lower flowrate range of 0 to 40 µL·min-1.

Computing Metal-Binding Proteins for Therapeutic Benefit.

Over one third of biomolecules rely on metal ions to exert their cellular functions. Metal ions can play a structural role by stabilizing the structure of biomolecules, a functional role by promoting a wide variety of biochemical reactions, and a regulatory role by acting as messengers upon binding to proteins regulating cellular metal-homeostasis. These diverse roles in biology ascribe critical implications to metal-binding proteins in the onset of many diseases. Hence, it is of utmost importance to exhaustively unlock the different mechanistic facets of metal-binding proteins and to harness this knowledge to rationally devise novel therapeutic strategies to prevent or cure pathological states associated with metal-dependent cellular dysfunctions. In this compendium, we illustrate how the use of a computational arsenal based on docking, classical, and quantum-classical molecular dynamics simulations can contribute to extricate the minutiae of the catalytic, transport, and inhibition mechanisms of metal-binding proteins at the atomic level. This knowledge represents a fertile ground and an essential prerequisite for selectively targeting metal-binding proteins with small-molecule inhibitors aiming to (i) abrogate deregulated metal-dependent (mis)functions or (ii) leverage metal-dyshomeostasis to selectively trigger harmful cells death.

Cu(I) Controls Conformational States in Human Atox1 Metallochaperone: An EPR and Multiscale Simulation Study.

Atox1 is a human copper metallochaperone that is responsible for transferring copper ions from the main human copper transporter, hCtr1, to ATP7A/B in the Golgi apparatus. Atox1 interacts with the Ctr1 C-terminal domain as a dimer, although it transfers the copper ions to ATP7A/B in a monomeric form. The copper binding site in the Atox1 dimer involves Cys12 and Cys15, while Lys60 was also suggested to play a role in the copper binding. We recently showed that Atox1 can adopt various conformational states, depending on the interacting protein. In the current study, we apply EPR experiments together with hybrid quantum mechanics-molecular mechanics molecular dynamics simulations using a recently developed semiempirical density functional theory approach, to better understand the effect of Atox1's conformational states on copper coordination. We propose that the flexibility of Atox1 occurs owing to protonation of one or more of the cysteine residues, and that Cys15 is an important residue for Atox1 dimerization, while Cys12 is a critical residue for Cu(I) binding. We also show that Lys60 electrostatically stabilizes the Cu(I)-Atox1 dimer.

Structural, Thermodynamic, and Kinetic Traits of Antiestrogen-Compounds Selectively Targeting the Y537S Mutant Estrogen Receptor α Transcriptional Activity in Breast Cancer Cell Lines.

The most frequently diagnosed cancers in women are the estrogen receptor (ER)-positive breast cancer subtypes, which are characterized by estrogen dependency for their growth. The mainstay of clinical treatment for this tumor relies on the modulation of ERα action or on the suppression of estrogen biosynthesis via the administration of Selective ERα Modulators/Down-regulators (SERMs/SERDs) or aromatase inhibitors, respectively. Nevertheless, de novo and acquired resistance to these therapies frequently occurs and represents a major clinical concern for patient survival. Recently, somatic mutations affecting the hormone-binding domain of ERα (i.e., Y537S, Y537N, D538G) have been associated with endocrine resistance, disease relapse and increased mortality rates. Hence, devising novel therapies against these ERα isoforms represents a daunting challenge. Here, we identified five molecules active on recurrent Y537S ERα polymorphism by employing in silico virtual screening on commercial databases of molecules, complemented by ER-transactivation and MTT assays in MCF7 and MDA-MB-231 breast cancer cells expressing wild type or mutated ERα. Among them, one molecule selectively targets Y537S ERα without inducing any cytotoxicity in breast cell lines. Multi-microseconds (4.5 µs) of biased and unbiased molecular dynamics provided an atomic-level picture of the structural, thermodynamics (i.e., binding free energies) and the kinetic (i.e., dissociation free energy barriers) of these active ligands as compared to clinically used SERM/SERDs upon binding to wild type and distinct ERα variants (Y537S, Y537N, D538G). This study contributes to a dissection of the key molecular traits needed by drug-candidates to hamper the agonist (active)-like conformation of ERα, normally selected by those polymorphic variants. This information can be useful to discover mutant specific drug-candidates, enabling to move a step forward toward tailored approaches for breast cancer treatment.

Unraveling the Impact of Cysteine-to-Serine Mutations on the Structural and Functional Properties of Cu(I)-Binding Proteins.

Appropriate maintenance of Cu(I) homeostasis is an essential requirement for proper cell function because its misregulation induces the onset of major human diseases and mortality. For this reason, several research efforts have been devoted to dissecting the inner working mechanism of Cu(I)-binding proteins and transporters. A commonly adopted strategy relies on mutations of cysteine residues, for which Cu(I) has an exquisite complementarity, to serines. Nevertheless, in spite of the similarity between these two amino acids, the structural and functional impact of serine mutations on Cu(I)-binding biomolecules remains unclear. Here, we applied various biochemical and biophysical methods, together with all-atom simulations, to investigate the effect of these mutations on the stability, structure, and aggregation propensity of Cu(I)-binding proteins, as well as their interaction with specific partner proteins. Among Cu(I)-binding biomolecules, we focused on the eukaryotic Atox1-ATP7B system, and the prokaryotic CueR metalloregulator. Our results reveal that proteins containing cysteine-to-serine mutations can still bind Cu(I) ions; however, this alters their stability and aggregation propensity. These results contribute to deciphering the critical biological principles underlying the regulatory mechanism of the in-cell Cu(I) concentration, and provide a basis for interpreting future studies that will take advantage of cysteine-to-serine mutations in Cu(I)-binding systems.

Copper trafficking in eukaryotic systems: current knowledge from experimental and computational efforts.

Copper plays a vital role in fundamental cellular functions, and its concentration in the cell must be tightly regulated, as dysfunction of copper homeostasis is linked to severe neurological diseases and cancer. This review provides a compendium of current knowledge regarding the mechanism of copper transfer from the blood system to the Golgi apparatus; this mechanism involves the copper transporter hCtr1, the metallochaperone Atox1, and the ATPases ATP7A/B. We discuss key insights regarding the structural and functional properties of the hCtr1-Atox1-ATP7B cycle, obtained from diverse studies relying on distinct yet complementary biophysical, biochemical, and computational methods. We further address the mechanistic aspects of the cycle that continue to remain elusive. These knowledge gaps must be filled in order to be able to harness our understanding of copper transfer to develop therapeutic approaches with the capacity to modulate copper metabolism.

The pivotal role of MBD4-ATP7B in the human Cu(i) excretion path as revealed by EPR experiments and all-atom simulations.

Copper's essentiality and toxicity require a meticulous mechanism for its acquisition, cellular distribution and excretion, which remains hitherto elusive. Herein, we jointly employed electron paramagnetic resonance spectroscopy and all-atom simulations to resolve the copper trafficking mechanism in humans considering the route travelled by Cu(i) from the metallochaperone Atox1 to the metal binding domains 3 and 4 of ATP7B. Our study shows that Cu(i) in the final part of its extraction pathway is most likely mediated by binding of Atox1 monomer to MBD4 of ATP7B. This interaction takes place through weak metal-stabilized protein-protein interactions.

Rational design of allosteric modulators of the aromatase enzyme: An unprecedented therapeutic strategy to fight breast cancer.

Estrogens play a key role in cellular proliferation of estrogen-receptor-positive (ER+) breast cancers (BCs). Suppression of estrogen production by competitive inhibitors of the enzyme aromatase (AIs) is currently one of the most effective therapies against ER + BC. Yet, the development of acquired resistance, after prolonged treatments with AIs, represents a clinical major concern. Serendipitous findings indicate that aromatase may be non-competitively inhibited by clinically employed drugs and/or industrial chemicals. Here, by performing in silico screening on two putative allosteric sites, molecular dynamics and free energy simulations, supported by enzymatic and cell-based assays, we identified five leads inhibiting the enzyme via a non-active site-directed mechanism. This study provides new compelling evidences for the existence of an allosteric regulation of aromatase and for the possibility of exploiting it to modulate estrogens biosynthesis. Such modulation can aptly reduce side effects caused by the complete estrogen deprivation therapy, and, possibly, delay/avoid the onset of resistance.

A Dehydrogenase Dual Hydrogen Abstraction Mechanism Promotes Estrogen Biosynthesis: Can We Expand the Functional Annotation of the Aromatase Enzyme?

Cytochrome P450 (CYP450) enzymes are involved in the metabolism of exogenous compounds and in the synthesis of signaling molecules. Among the latter, human aromatase (HA) promotes estrogen biosynthesis, which is a key pharmacological target against breast cancers. After decades of debate, interest in gaining a comprehensive picture of HA catalysis has been renewed by the recent discovery that compound I (Cpd I) is the reactive species of the peculiar aromatization step. Herein, for the first time, a complete atomic-level picture of all controversial steps of estrogen biosynthesis is presented. By performing cumulative quantum-classical molecular dynamics and metadynamics simulations of about 180 ps, it is revealed that the most likely enzymatic path relies on three factors: 1) androstenedione enolization and compound 0 (Cpd 0) formation through a proton network mediated by Asp309; 2) subsequent formation of Cpd I, upon rearrangement of the Asp309 side chain and the establishment of a proton network involving Asp309 and Thr310; and 3) after two hydroxylation reactions, 19,19-gem-diol is converted into estrone by Cpd I, through an uncommon dehydrogenase-like dual hydrogen abstraction mechanism. As a result, HA performs estrogen biosynthesis by merging hydroxylase with dehydrogenase activity, which suggests that the need to perform complex chemical transformations led nature to engineer HA, and possibly other steroidogenic CYP450s, by expanding its range of functions to achieve an optimal catalytic efficiency.

A Computational Assay of Estrogen Receptor α Antagonists Reveals the Key Common Structural Traits of Drugs Effectively Fighting Refractory Breast Cancers.

Somatic mutations of the Estrogen Receptor α (ERα) occur with an up to 40% incidence in ER sensitive breast cancer (BC) patients undergoing prolonged endocrine treatments. These polymorphisms are implicated in acquired resistance, disease relapse, and increased mortality rates, hence representing a current major clinical challenge. Here, multi-microseconds (12.5 µs) molecular dynamics simulations revealed that recurrent ERα polymorphisms (i. e. L536Q, Y537S, Y537N, D538G) (mERα) are constitutively active in their apo form and that they prompt the selection of an agonist (active)-like conformation even upon antagonists binding. Interestingly, our simulations rationalize, for the first time, the efficacy profile of (pre)clinically used Selective Estrogen Receptor Modulators/Downregulators (SERMs/SERDs) against these variants, enlightening, at atomistic level of detail, the key common structural traits needed by drugs able to effectively fight refractory BC types. This knowledge represents a key advancement for mechanism-based therapeutics targeting resistant ERα isoforms, potentially allowing the community to move a step closer to 'precision medicine' calibrated on patients' genetic profiles and disease progression.

Carnosine and Homocarnosine Degradation Mechanisms by the Human Carnosinase Enzyme CN1: Insights from Multiscale Simulations.

The endogenous dipeptide l-carnosine, and its derivative homocarnosine, prevent and reduce several pathologies like amytrophic lateral sclerosis (ALS), Alzheimer's disease, and Parkinson's disease. Their beneficial action is severely hampered because of the hydrolysis by carnosinase enzymes, in particular the human carnosinase, hCN1. This belongs to the metallopeptidase M20 family, where a cocatalytic active site is formed by two Zn(2+) ions, bridged by a hydroxide anion. The protein may exist as a monomer and as a dimer in vivo. Here we used hybrid quantum mechanics/molecular mechanics simulations based on the dimeric apoenzyme's structural information to predict the Michaelis complexes with l-carnosine and its derivative homocarnosine. On the basis of our calculations, we suggest that (i) l-carnosine degradation occurs through a nucleophilic attack of a Zn(2+)-coordinated bridging moiety for both monomer and dimer. This mechanistic hypothesis for hCN1 catalysis differs from previous proposals, while it is in agreement with available experimental data. (ii) The experimentally measured higher affinity of homocarnosine for the enzyme relative to l-carnosine might be explained, at least in part, by more extensive interactions inside the monomeric and dimeric hCN1's active site. (iii) Hydrogen bonds at the binding site, present in the dimer but absent in the monomer, might play a role in the experimentally observed higher activity of the dimeric form. Investigations of the enzymatic reaction are required to establish or disprove this hypothesis. Our results may serve as a basis for the design of potent hCN1 inhibitors.

The Chemistry of Neurodegeneration: Kinetic Data and Their Implications.

We collected experimental kinetic rate constants for chemical processes responsible for the development and progress of neurodegeneration, focused on the enzymatic and non-enzymatic degradation of amine neurotransmitters and their reactive and neurotoxic metabolites. A gross scheme of neurodegeneration on the molecular level is based on two pathways. Firstly, reactive species oxidise heavy atom ions, which enhances the interaction with alpha-synuclein, thus promoting its folding to the beta form and giving rise to insoluble amyloid plaques. The latter prevents the function of vesicular transport leading to gradual neuronal death. In the second pathway, radical species, OH(·) in particular, react with the methylene groups of the apolar part of the lipid bilayer of either the cell or mitochondrial wall, resulting in membrane leakage followed by dyshomeostasis, loss of resting potential and neuron death. Unlike all other central neural system (CNS)-relevant biogenic amines, dopamine and noradrenaline are capable of a non-enzymatic auto-oxidative reaction, which produces hydrogen peroxide. This reaction is not limited to the mitochondrial membrane where scavenging enzymes, such as catalase, are located. On the other hand, dopamine and its metabolites, such as dopamine-o-quinone, dopaminechrome, 5,6-dihydroxyindole and indo-5,6-quinone, also interact directly with alpha-synuclein and reversibly inhibit plaque formation. We consider the role of the heavy metal ions, selected scavengers and scavenging enzymes, and discuss the relevance of certain foods and food supplements, including curcumin, garlic, N-acetyl cysteine, caffeine and red wine, as well as the long-term administration of non-steroid anti-inflammatory drugs and occasional tobacco smoking, that could all act toward preventing neurodegeneration. The current analysis can be employed in developing strategies for the prevention and treatment of neurodegeneration, and, hopefully, aid in the building of an overall kinetic molecular model of neurodegeneration itself.

Quantum-chemical approach to determining the high potency of clorgyline as an irreversible acetylenic monoamine oxidase inhibitor.

Density functional theory calculations were employed to investigate the nature of chemical bond formation between the flavin co-factor of the enzyme monoamine oxidase (MAO) and its irreversible acetylenic inhibitor clorgyline in its terminally deprotonated anionic form. Since MAOs regulate the level of neurotransmitters in living cells, this reaction is pharmacologically relevant for treating depression and other mood disorders. The results revealed that this pathway is associated with the activation free energy of ΔG act (#) = 17.4 kcal mol(-1), which, together with our previous results, suggests that clorgyline is intrinsically a more effective MAO inhibitor than antiparkinsonian drugs rasagiline and selegiline considering the preferred MAO isoforms in each case, thus displaying a trend in agreement with experimental data. The reaction is facilitated by the pronounced electrophilic character of the flavin moiety, due to its ability to efficiently accommodate excess negative charge from the approaching anionic inhibitor through resonance effect. The investigated mechanism was additionally validated by the inspection of the geometry of the flavin moiety in the formed adduct, which exhibit distortion from planarity consistent with experimental observations. These results offer valuable insight for mechanistic studies on other flavoenzymes and for the design of new antidepressants and antiparkinsonian drugs.