Detecting DNA Mismatches with Metallo-Insertors: A Molecular Simulation Study

Molecules that selectively recognize DNA mismatches (MMs) play a key role as nucleic acids probes and as chemotherapeutic agents. Metallo-insertors bind to the minor groove (mG) of double strand (ds) DNA, expelling the mismatched base pairs and acting as their pi-stacking replacement. In contrast, metallo-intercalators bind to the major groove (MG) of ds DNA and pi-stack to adjacent base pairs. In this study we focused on structural and energetic properties of Delta-[Rh(bpy)(2)(chrysi)](3+) (1), Delta-[Ru(bpy)(2)(ddpz)](2+) (2), and Delta-[Ru(bpy)(2)(eilatin)](2+) (3) as prototypical examples of metallo-insertors and intercalators. For all molecules we characterized both insertion and intercalation into a DNA dodecamer via force field based molecular dynamics (MD) and hybrid quantum-classical (QM/MM) MD simulations. A structural analysis of the 1-3/DNA noncovalent adducts reveals that the insertion provokes an untwist of the DNA, an opening of the mG and of the phosphate backbone in proximity of the mismatch, while the intercalation induces smaller changes of these structural parameters. This behavior appears to be correlated with the size of the inserting/intercalating ligand in proximity of the metal coordination site. Moreover, our simulations show that the different selectivity of 1 toward distinct MM types may be correlated with the thermodynamic stability of the MMs in the free DNA and with that of the corresponding insertion adduct. Understanding the factors which tune a specific insertion is of crucial importance for designing specific luminescent probes that selectively recognize MMs, as well as for developing more effective anticancer drugs active in MM repair of deficient cells lines

The catalytic mechanism of steroidogenic cytochromes P450 from all-atom simulations: Entwinement with membrane environment, redox partners, and post-transcriptional regulation

Cytochromes P450 (CYP450s) promote the biosynthesis of steroid hormones with major impact on the onset of diseases such as breast and prostate cancers. By merging distinct functions into the same catalytic scaffold, steroidogenic CYP450s enhance complex chemical transformations with extreme efficiency and selectivity. Mammalian CYP450s and their redox partners are membrane-anchored proteins, dynamically associating to form functional machineries. Mounting evidence signifies that environmental factors are strictly intertwined with CYP450s catalysis. Atomic-level simulations have the potential to provide insights into the catalytic mechanism of steroidogenic CYP450s and on its regulation by environmental factors, furnishing information often inaccessible to experimental means. In this review, after an introduction of computational methods commonly employed to tackle these systems, we report the current knowledge on three steroidogenic CYP450s\u2014CYP11A1, CYP17A1, and CYP19A1\u2014endowed with multiple catalytic functions and critically involved in cancer onset. In particular, besides discussing their catalytic mechanisms, we highlight how the membrane environment contributes to (i) regulate ligand channeling through these enzymes, (ii) modulate their interactions with specific protein partners, (iii) mediate post-transcriptional regulation induced by phosphorylation. The results presented set the basis for developing novel therapeutic strategies aimed at fighting diseases originating from steroid metabolism dysfunction

Investigating the molecular mechanism of h3b‐8800: A splicing modulator inducing preferential lethality in spliceosome‐mutant cancers

The SF3B1 protein, part of the SF3b complex, recognizes the intron branch point sequence of precursor messenger RNA (pre‐mRNA), thus contributing to splicing fidelity. SF3B1 is frequently mutated in cancer and is the target of distinct families of splicing modulators (SMs). Among these, H3B‐8800 is of particular interest, as it induces preferential lethality in cancer cells bearing the frequent and highly pathogenic K700E SF3B1 mutation. Despite the potential of H3B‐8800 to treat myeloid leukemia and other cancer types hallmarked by SF3B1 mutations, the molecular mechanism underlying its preferential lethality towards spliceosome‐mutant cancer cells remains elusive. Here, microsecond‐long all‐atom simulations addressed the binding/dissociation mechanism of H3B‐8800 to wild type and K700E SF3B1‐containing SF3b (K700ESB3b) complexes at the atomic level, unlocking that the K700E mutation little affects the thermodynamics and kinetic traits of H3B‐8800 binding. This supports the hypothesis that the selectivity of H3B‐8800 towards mutant cancer cells is unrelated to its preferential targeting ofK700ESB3b. Nevertheless, this set of simulations discloses that the K700E mutation and H3B‐8800 binding affect the overall SF3b internal motion, which in turn may influence the way SF3b interacts with other spliceosome components. Finally, we unveil the existence of a putative druggable SF3b pocket in the vicinity of K700E that could be harnessed in future rational drug‐discovery efforts to specifically target mutant SF3b

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

Enantioselective palladium-catalyzed hydrosilylation of styrene: Detailed reaction mechanism from first-principles and hybrid QM/MM molecular dynamics simulations

The mechanism of the enantioselective hydrosilylation of styrene catalyzed by Pd-0 species generated in situ from dichloro {1-{(R)-1-[(S)-2(diphenylphosphino-kappaP)ferrocenyl]ethyl}-3-trimethylphenyl-5-1H-pyrazole-kappaN}palladium, 1, has been investigated in detail through ab initio molecular dynamics and hybrid ab initio molecular dynamics/molecular mechanics (QM/MM) calculations. Different QM/MM models have been adopted in order to probe the specific steric and electronic contributions of different substituents. The catalytic cycle is initiated by the formation of a weakly bound pi-complex (DeltaE approximate to -5.4 kcal/mol) under simultaneous detachment of the pyrazole ligand. In agreement with a Chalk-Harrod mechanism, this is followed by the migratory insertion of the hydride, which leads to a eta(3)-coordination mode of the benzylic fragment. The significant stabilization of the allylic intermediate (DeltaE approximate to -11 kcal/mol) is responsible for the high regioselectivity of the reaction (as well as for its enantioselectivity). The rate-determining step with an activation barrier of 16 kcal/mol is the migration of the silyl ligand to the a-carbon of the substrate with concomitant closure of the ligand chelate ring. This step leads to the formation of an intermediate in which the phenyl moiety of the product remains coordinated in an eta(2)-mode to the palladium. The addition of trichlorosilane leads to product formation and hence to the regeneration of the catalyst. A unimolecular reaction pathway on the other hand, in which the transfer of the silyl ligand to the benzylic fragment is concerted with the addition of a molecule of HSiCl3 to the catalyst, is disfavored by an activation barrier of similar to30 kcal/mol

Metadynamics Simulations Reveal a Na+ Independent Exiting Path of Galactose for the Inward-Facing Conformation of vSGLT

Sodium-Galactose Transporter (SGLT) is a secondary active symporter which accumulates sugars into cells by using the electrochemical gradient of Na+ across the membrane. Previous computational studies provided insights into the release process of the two ligands (galactose and sodium ion) into the cytoplasm from the inward-facing conformation of Vibrio parahaemolyticus sodium/galactose transporter (vSGLT). Several aspects of the transport mechanism of this symporter remain to be clarified: (i) a detailed kinetic and thermodynamic characterization of the exit path of the two ligands is still lacking; (ii) contradictory conclusions have been drawn concerning the gating role of Y263; (iii) the role of Na+ in modulating the release path of galactose is not clear. In this work, we use bias-exchange metadynamics simulations to characterize the free energy profile of the galactose and Na+ release processes toward the intracellular side. Surprisingly, we find that the exit of Na+ and galactose is non-concerted as the cooperativity between the two ligands is associated to a transition that is not rate limiting. The dissociation barriers are of the order of 11-12 kcal/mol for both the ion and the substrate, in line with kinetic information concerning this type of transporters. On the basis of these results we propose a branched six-state alternating access mechanism, which may be shared also by other members of the LeuT-fold transporters

The hydrolysis mechanism of the anticancer ruthenium drugs NAMI-A and ICR investigated by DFT-PCM calculations

(ImH)[trans-RuCl4(DMSO-S)(Im)], (Im = imidazole, DMSO-S = S-bonded dimethylsulfoxide), NAMI-A, is the first anticancer ruthenium compound that successfully completed Phase I clinical trials. NAMI-A shows a remarkable activity against lung metastases of solid tumors, but is not effective in the reduction of primary cancer. The structurally similar (ImH)[trans-RuCl4(Im)(2)], ICR (or KP418), and its indazole analog (KP1019) are promising candidate drugs in the treatment of colorectal cancers, but have no antimetastatic activity. Despite the pharmacological relevance of these compounds, no rationale has been furnished to explain their markedly different activity. While the nature of the chemical species responsible for their antimetastatic/anticancer activity has not been determined, it has been suggested that the difference between reduction potentials of NAMI-A and ICR may be the key to the different biological responses they induce. In this work, Density Functional Theory calculations were performed to investigate the hydrolysis of NAMI-A and ICR in both Ru-III and Ru-II oxidation states, up to the third aquation. In line with experimental findings, our calculations provide a picture of the hydrolysis of NAMI-A and ICR mainly as a stepwise loss of chloride ligands. While dissociation of Im is unlikely under neutral conditions, that of DMSO becomes competitive with the loss of chloride ions as the hydrolysis proceeds. Redox properties of NAMI-A and ICR and of their most relevant hydrolytic intermediates were also studied in order to monitor the effects of biological reductants on the mechanism of action. Our findings may contribute to the identification of the active compounds that interact with biological targets, and to explain the different biological activity of NAMI-A and ICR

Disclosing the impact of carcinogenic SF3b mutations on pre-mRNA recognition via all-atom simulations

The spliceosome accurately promotes precursor messenger-RNA splicing by recognizing specific noncoding intronic tracts including the branch point sequence (BPS) and the 3'-splice-site (3\u2018SS). Mutations of Hsh155 (yeast)/SF3B1 (human), which is a protein of the SF3b factor involved in BPS recognition and induces altered BPS binding and 3\u2018SS selection, lead to mis-spliced mRNA transcripts. Although these mutations recur in hematologic malignancies, the mechanism by which they change gene expression remains unclear. In this study, multi-microsecond-long moleculardynamics simulations of eighth distinct ~700,000 atom models of the spliceosome Bact complex, and gene sequencing of SF3B1, disclose that these carcinogenic isoforms destabilize intron binding and/or affect the functional dynamics of Hsh155/SF3B1 only when binding non-consensus BPSs, as opposed to the non-pathogenic variants newly annotated here. This pinpoints a cross-talk between the distal Hsh155 mutation and BPS recognition sites. Our outcomes unprecedentedly contribute to elucidating the principles of pre-mRNA recognition, which provides critical insights on the mechanism underlying constitutive/alternative/aberrant splicing

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

Switching from Aromatase Inhibitors to Dual Targeting Flavonoid-Based Compounds for Breast Cancer Treatment

Despite the significant outcomes attained by scientific research, breast cancer (BC) still represents the second leading cause of death in women. Estrogen receptor-positive (ER+) BC accounts for the majority of diagnosed BCs, highlighting the disruption of estrogenic signalling as target for first-line treatment. This goal is presently pursued by inhibiting aromatase (AR) enzyme or by modulating Estrogen Receptor (ER) α. An appealing strategy for fighting BC and reducing side effects and resistance issues may lie in the design of multifunctional compounds able to simultaneously target AR and ER. In this paper, previously reported flavonoid-related potent AR inhibitors were suitably modified with the aim of also targeting ERα. As a result, homoisoflavone derivatives 3b and 4a emerged as well-balanced submicromolar dual acting compounds. An extensive computational study was then performed to gain insights into the interactions the best compounds established with the two targets. This study highlighted the feasibility of switching from single-target compounds to balanced dual-acting agents, confirming that a multi-target approach may represent a valid therapeutic option to counteract ER+ BC. The homoisoflavone core emerged as a valuable natural-inspired scaffold for the design of multifunctional compounds