A Comprehensive Computational Insight into the PD-L1 Binding to PD-1 and Small Molecules.

Immunotherapy has marked a revolution in cancer therapy. The most extensively studied target in this field is represented by the protein-protein interaction between PD-1 and its ligand, PD-L1. The promising results obtained with the clinical use of monoclonal antibodies (mAbs) directed against both PD-1 and PD-L1 have prompted the search for small-molecule binders capable of disrupting the protein-protein contact and overcoming the limitations presented by mAbs. The disclosure of the first X-ray complexes of PD-L1 with BMS ligands showed the protein in dimeric form, with the ligand in a symmetrical hydrophobic tunnel. These findings paved the way for the discovery of new ligands. To this end, and to understand the binding mechanism of small molecules to PD-L1 along with the dimerization process, many structure-based computational studies have been applied. In the present review, we examined the most relevant articles presenting computational analyses aimed at elucidating the binding mechanism of PD-L1 with PD-1 and small molecule ligands. Additionally, virtual screening studies that identified validated PD-L1 ligands were included. The relevance of the reported studies highlights the increasingly prominent role that these techniques can play in chemical biology and drug discovery.

A computational insight on the aromatic amino acids conjugation with [Cp*Rh(H2O)3]2+ by using the meta-dynamics/FMO3 approach.

CONTEXT: Rh(III) complexes demonstrated to exert promising pharmacological effects with potential applications as anti-cancer, anti-bacterial, and antimicrobial agents. One important Rh(III)-ligand is the pentamethylcyclopentadienyl (Cp*) group forming in water the [Cp*Rh(H2O)3]2+ complex. Among of its attractive chemical properties is the ability to react specifically with Tyr amino acid side chain of G-protein-coupled receptor (GPCR) peptides by means of highly chemoselective bioconjugation reaction, at room temperature and at pH 5-6. In this computational work, in order to deepen the mechanism of this chemoselective conjugation, we study the ligand exchange reaction between [Cp*Rh(H2O)3]2+ and three small molecules, namely p-cresol, 3-methylimidazole, and toluene, selected as mimetic of aromatic side chains of tyrosine (Tyr), tryptophan (Trp) and phenylalanine (Phe), respectively. Our outcomes suggest that the high selectivity for Tyr side chain might be related to OH group able to affect both thermodynamic and kinetic of ligand exchange reaction, due to its ability to act as both H bond acceptor and donor. These mechanistic aspects can be used to design new metal drugs containing the [Cp*Rh]2+ scaffold targeting specifically Tyr residues involved in biological/pathological processes such as phosphorylation by means of Tyr-kinase enzyme and protein-protein interactions. METHODS: The geometry of three encounter complexes and product adducts were optimized at the B3LYP//CPCM/ωB97X-D level of theory, adopting the 6-311+G(d,p) basis set for all non-metal atoms and the LANL2DZ pseudopotential for the Rh atom. Meta-dynamics RMSD (MTD(RMSD)) calculations at GFN2-xTB level of theory were performed in NVT conditions at 298.15 K to investigate the bioconjugation reactions (simulation time: 100 ps; integration step 2.0; implicit solvent model: GBSA). The MTD(RMSD) simulation was performed in two replicates for each encounter complex. Final representative subsets of 100 structures for each run were gained with a sampling rate of 1 ps and analyzed by performing single point calculations using the FMO3 method at RI-MP2/6-311G//PCM[1] level of theory, adopting the MCP-TZP core potential for Rh atom.

IRMPD spectroscopy and quantum-chemical simulations of the reaction products of cisplatin with the dipeptide CysGly.

The inorganic antineoplastic drug cisplatin was made to react in solution with the dipeptide cysteinylglycine (CysGly), chosen as a functional model of glutathione, and the reaction products were analyzed using electrospray ionization mass spectrometry (ESI-MS). Selected complexes, i.e., the primary substitution product cis-[PtCl(NH3)2(CysGly)]+ and the chelate cis-[PtCl(NH3)(CysGly)]+, were submitted to IR multiple photon dissociation (IRMPD) spectroscopy obtaining their vibrational features. The experimental IR ion spectra were compared with the calculated IR absorptions of different plausible isomeric families, finding CysGly to bind preferentially platinum(II) via its deprotonated thiolic group in the monovalent complex, cis-[PtCl(NH3)2(CysGly)]+, and to evolve in the S,N-bound chelate structure cis-[PtCl(NH3)(CysGly)]+ through the SH and NH2 functionality of the cysteine residue. Moreover, our findings indicate that the platination reaction does not affect the CysGly peptide bond, which remains in its trans configuration. These results provide additional insights into the reactivity of Pt(II)-complexes with glutathione which is involved in cellular cisplatin resistance.

The FMO2 analysis of the ligand-receptor binding energy: the Biscarbene-Gold(I)/DNA G-Quadruplex case study.

In this work, the ab initio fragment molecular orbital (FMO) method was applied to calculate and analyze the binding energy of two biscarbene-Au(I) derivatives, [Au(9-methylcaffein-8-ylidene)2]+ and [Au(1,3-dimethylbenzimidazol-2-ylidene)2]+, to the DNA G-Quadruplex structure. The FMO2 binding energy considers the ligand-receptor complex as well as the isolated forms of energy-minimum state of ligand and receptor, providing a better description of ligand-receptor affinity compared with simple pair interaction energies (PIE). Our results highlight important features of the binding process of biscarbene-Au(I) derivatives to DNA G-Quadruplex, indicating that the total deformation-polarization energy and desolvation penalty of the ligands are the main terms destabilizing the binding. The pair interaction energy decomposition analysis (PIEDA) between ligand and nucleobases suggest that the main interaction terms are electrostatic and charge-transfer energies supporting the hypothesis that Au(I) ion can be involved in π-cation interactions further stabilizing the ligand-receptor complex. Moreover, the presence of polar groups on the carbene ring, as C = O, can improve the charge-transfer interaction with K+ ion. These findings can be employed to design new powerful biscarbene-Au(I) DNA-G quadruplex binders as promising anticancer drugs. The procedure described in this work can be applied to investigate any ligand-receptor system and is particularly useful when the binding process is strongly characterized by polarization, charge-transfer and dispersion interactions, properly evaluated by ab initio methods.

Elusive intermediates in cisplatin reaction with target amino acids: Platinum(II)-cysteine complexes assayed by IR ion spectroscopy and DFT calculations.

The reactivity of a widely used metal based antineoplastic drug, cisplatin, cis-PtCl2(NH3)2, with L-cysteine (Cys) has been investigated using a combination of electrospray ionization mass spectrometry (ESI-MS), IRMPD gas phase ion spectroscopy and DFT calculations. The cysteine lateral chain represents one of the main platination sites in proteins, which is believed to be related to the resistance mechanisms to cisplatin. The vibrational features of the mass-selected substitution product cis-[PtCl(NH3)2(Cys)]+ and the intercepted cis-[PtCl(NH3)2(H2O)(Cys)]+ intermediate complex were compared to calculated IR spectra, enabling the assessment of the sampled ions structures. In cis-[PtCl(NH3)2(Cys)]+, cysteine was found to bind platinum through the sulfur atom as a thiolate zwitterion, highlighting the enhanced acidity of the cysteine thiol group upon metal coordination. The cis-[PtCl(NH3)2(H2O)(Cys)]+ structure complies with the non-covalent encounter complex, formed by cis-[PtCl(NH3)2(H2O)]+ and neutral cysteine. This species is able to undergo the substitution process to produce cis-[PtCl(NH3)2(Cys)]+ when activated as a mass-isolated ion suggesting its participation in the reaction mechanism of cisplatin with cysteine in solution. Finally, the DFT-calculated energy profile for the substitution reaction was correlated with the peculiar gas-phase reactivity of this non-covalent complex, resulting to be 10-fold less reactive toward substitution than the corresponding methionine complex.

Homodimeric complexes of the 90-231 human prion: a multilayered computational study based on FMO/GRID-DRY approach.

The molecular interaction properties and aggregation capabilities disclosed by PrP-E200K, a pathogenic mutant of the human prion protein, were investigated in detail using multilayered computational approaches. In a previous work, we reported that the electrostatic complementarity between region1 (negative) and region3 (positive) has been assumed to lead to a head-to tail interaction between 120 and 231 PrP-E200K units and to initiation of the aggregation process. In this work, we extended the PrP-E200K structure by including the unstructured 90-120 segment which was found to assume different conformations. Plausible models of 90-231 PrP-E200K dimers were calculated and analyzed in depth to identify the nature of the involved protein-protein interactions. The unstructured 90-120 segment was found to extend the positively charged region3 involved in the association of PrP-E200K units which resulted to be driven by hydrophobic interactions. The combination of molecular dynamics, protein-protein docking, grid-based mapping, and fragment molecular orbital approaches allowed us to provide a plausible mechanism of the early state of 90-231 PrP-E200K aggregation, considered a preliminary step of amyloid conversion.

IRMPD Spectroscopy of Bare Monodeprotonated Genistein, an Antioxidant Flavonoid.

Genistein is a naturally occurring polyphenol belonging to the family of flavonoids with estrogenic properties and proven antioxidant, anti-inflammatory, and hormonal effects. Genistein and its derivatives are involved in radical scavenging activity by way of mechanisms based on sequential proton-loss electron transfer. In view of this role, a detailed structural characterization of its bare deprotonated form, [geni-H]-, generated by electrospray ionization, has been performed by tandem mass spectrometry and infrared multiple photon dissociation (IRMPD) spectroscopy in the 800-1800 cm-1 spectral range. Quantum chemical calculations at the B3LYP/6-311+G(d,p) level of theory were carried out to determine geometries, thermochemical data, and anharmonic vibrational properties of low-lying isomers, enabling to interpret the experimental spectrum. Evidence is gathered that the conjugate base of genistein exists as a single isomeric form, which is deprotonated at the most acidic site (7-OH) and benefits from a strong intramolecular H-bond interaction between 5-OH and the adjacent carbonyl oxygen in the most stable arrangement.

From Preassociation to Chelation: A Survey of Cisplatin Interaction with Methionine at Molecular Level by IR Ion Spectroscopy and Computations.

Methionine (Met) plays an important role in the metabolism of cisplatin anticancer drug. Yet, methionine platination in aqueous solution presents a highly complex pattern of interconnected paths and intermediates. This study reports on the reaction of methionine with the active aqua form of cisplatin, cis-[PtCl(NH3)2(H2O)]+, isolating the encounter complex of the reactant pair, {cis-[PtCl(NH3)2(H2O)]+·Met}, by electrospray ionization. In the unsolvated state, charged intermediates are characterized for their structure and photofragmentation behavior by IR ion spectroscopy combined with quantum-chemical calculations, obtaining an outline of the cisplatin-methionine reaction at a molecular level. To summarize the major findings: (i) the {cis-[PtCl(NH3)2(H2O)]+·Met} encounter complex, lying on the reaction coordinate of the Eigen-Wilkins preassociation mechanism for ligand substitution, is delivered in the gas phase and characterized by IR ion spectroscopy; (ii) upon vibrational excitation, ligand exchange occurs within {cis-[PtCl(NH3)2(H2O)]+·Met}, releasing water and cis-[PtCl(NH3)2(Met)]+, along the calculated energy profile; (iii) activated cis-[PtCl(NH3)2(Met)]+ ions undergo NH3 departure, forming a chelate complex, [PtCl(NH3)(Met)]+, whose structure is congruent with overwhelming S-Met ligation as the primary coordination step. The latter process involving ammonia loss marks a difference with the prevailing chloride replacement in protic solvent, pointing to the effect of a low-polarity environment.

Hampering the early aggregation of PrP-E200K protein by charge-based inhibitors: a computational study.

A multilayered computational workflow was designed to identify a druggable binding site on the surface of the E200K pathogenic mutant of the human prion protein, and to investigate the effect of the binding of small molecules in the inhibition of the early aggregation of this protein. At this purpose, we developed an efficient computational tool to scan the molecular interaction properties of a whole MD trajectory, thus leading to the characterization of plausible binding regions on the surface of PrP-E200K. These structural data were then employed to drive structure-based virtual screening and fragment-based approaches to the seeking of small molecular binders of the PrP-E200K. Six promising compounds were identified, and their binding stabilities were assessed by MD simulations. Therefore, analyses of the molecular electrostatic potential similarity between the bound complexes and unbound protein evidenced their potential activity as charged-based inhibitors of the PrP-E200K early aggregation.

Dual Carbonic Anhydrase IX/XII Inhibitors and Carbon Monoxide Releasing Molecules Modulate LPS-Mediated Inflammation in Mouse Macrophages.

Low concentrations of carbon monoxide (CO) were reported to exhibit anti-inflammatory effects when administered in cells by suitable chemotypes such as CO releasing molecules (CO-RMs). In addition, the pH-modulating abilities of specific carbonic anhydrase isoforms played a crucial role in different models of inflammation and neuropathic pain. Herein, we report a series of chemical hybrids consisting of a Carbonic Anhydrase (CA) inhibitor linked to a CO-RM tail (CAI/CO-RMs). All compounds and their precursors were first tested in vitro for their inhibition activity against the human CA I, II, IX, and XII isoforms as well their CO releasing properties, aiming at corroborating the data by means of molecular modelling techniques. Then, their impact on metabolic activity modulation of RAW 264.7 mouse macrophages for 24 and 48 h was assessed with or without lipopolysaccharide (LPS) stimulation. The compounds were shown to counteract the inflammatory stimulus as also indicated by the reduced tumor necrosis factor alpha (TNF-α) release after treatment. All the biological results were compared to those of N-acetylcysteine (NAC) as a reference antioxidant compound. Within the series, two CAI/CO-RM hybrids (1 and 2), bearing both the well-known scaffold able to inhibit CAs (acesulfame) and the cobalt-based CO releasing portion, induced a higher anti-inflammatory effect up to 48 h at concentrations lower than NAC.

Reactions of cisplatin and cis-[PtI2(NH3)2] with molecular models of relevant protein sidechains: A comparative analysis.

Quite surprisingly, cisplatin and cis-[PtI2(NH3)2] were found to manifest significant differences in their reactions with the model protein lysozyme. We decided to explore whether these differences recur when reacting these two Pt compounds with other proteins. Notably, ESI-MS measurements carried out on cytochrome c nicely confirmed the reaction pattern observed for lysozyme. This prompted us to exploit a computational DFT approach to disclose the molecular basis of such behavior. We analyzed comparatively the reactions of cis-[PtCl2(NH3)2] and cis-[PtI2(NH3)2] with appropriate molecular models (Ls) of the sidechains of relevant aminoacids. We found that when Pt(II) complexes are reacted with sulfur ligands both quickly lose their halide ligands and then the resulting cis-[Pt(L)2(NH3)2] species loses ammonia upon reaction with a ligand excess. In the case of imidazole, again cis-[PtCl2(NH3)2] and cis-[PtI2(NH3)2] quickly lose their halide ligands but the resulting cis-[Pt(L)2(NH3)2] species does not lose ammonia by reaction with excess imidazole. These results imply that the two platinum complexes manifest a significantly different behavior in their reaction with representative small molecules in agreement with what observed in the case of model proteins. It follows that the protein itself must play a crucial role in triggering the peculiar reactivity of cis-[PtI2(NH3)2] and in governing the nature of the formed protein adducts. The probable reasons for the observed behavior are critically commented and discussed.

Characterization of PD-L1 binding sites by a combined FMO/GRID-DRY approach.

The programmed cell death protein 1 (PD-1) and its ligand, PD-L1, constitute an important co-inhibitory immune checkpoint leading to downregulation of immune system. Tumor cells developed a strategy to trigger PD-1/PD-L1 pathway reducing the T cell anticancer activity. Anti-PD-L1 small drugs, generally with improved pharmacokinetic and technological profiles than monoclonal antibodies, became an attractive research topic. Nevertheless, still few works have been published on the chemical features of possible binding sites. In this work, we applied a novel computational protocol based on the combination of the ab initio Fragment Molecular Orbital (FMO) method and a newly developed GRID-DRY approach in order to characterize the PD-L1 binding sites, starting from PD-1/PD-L1 and PD-L1/BMS-ligands (Bristol-Mayers Squibb ligands) complexes. The FMO method allows the calculation of the pair-residues as well as the ligand-residues interactions with ab initio accuracy, whereas the GRID-DRY approach is an effective tool to investigate hydrophobic interactions, not easily detectable by ab initio methods. The present GRID-DRY protocol is able to determine the energy contributions of each ligand atoms to each hydrophobic interaction, both qualitatively and quantitatively. We were also able to identify the three specific hot regions involved in PD-1/PD-L1 protein-protein interaction and in PD-L1/BMS-ligand interactions, in agreement with preceding theoretical/experimental results, and to suggest a specific pharmacophore for PD-L1 inhibitors.

An insight of early PrP-E200K aggregation by combined molecular dynamics/fragment molecular orbital approaches.

Unveiling the events leading to the formation of prion particles is a nowadays challenge in the field of neurochemistry. Pathogenic mutants of prion protein (PrP) are characterized by both an intrinsic tendency to aggregation and scrapie conversion propensity. However, the question about a possible correlation between these two events lasts still unanswered. Here, a multilayered computational workflow was employed to investigate structure, stability, and molecular interaction properties of a dimer of PrPC -E200K, a well-known mutant of the PrP that represents a reduced model of early aggregates of this protein. Based on the combination of molecular dynamics and quantum mechanical approaches, this study provided for an in depth insight of PrPC -E200K dimer in terms of residue-residue interactions. Assembly hypotheses for the early aggregation of PrPC -E200K are paved and compared with PrPSc models reported in the literature to find a structural link between early and late (scrapie) aggregates of this protein.

Cisplatin and transplatin interaction with methionine: bonding motifs assayed by vibrational spectroscopy in the isolated ionic complexes.

Cisplatin and transplatin (cis- and trans-[PtCl2(NH3)2]) have been allowed to react with methionine (Met) in water solution in a study aimed to characterize the monofunctional complex primarily formed. The thioether function of methionine is known to have a very high affinity for square planar platinum(ii) and sulfur-containing biomolecules have been proposed as a cisplatin drug reservoir on the way to platination at DNA. Both cisplatin and transplatin yield [PtCl(NH3)2Met]+ complexes, delivered by electrospray ionization in the gas phase and sampled as isolated species using tools based on mass spectrometry. The collision induced dissociation spectra of both cis-[PtCl(NH3)2Met]+ and trans-[PtCl(NH3)2Met]+ are quite similar and also the transport properties assayed by ion mobility mass spectrometry do not allow any appreciable discrimination. However, the vibrational spectra obtained by IR multiple photon absorption (IRMPD) spectroscopy show distinct features. Their analysis, supported by quantum chemical calculations, has revealed that while cisplatin attack is mainly directed to the sulfur atom of Met, transplatin shows a more balanced partition between sulfur and nitrogen binding. Among the vibrational signatures characterizing cis-[PtCl(NH3)2Met]+ and trans-[PtCl(NH3)2Met]+ complexes, the asymmetric NH2 stretching of the α-amino group of the amino acid at ca. 3440 cm-1 is peculiar and diagnostic of S-platination. IRMPD kinetics evaluated at this frequency support the prevailing S-attack by cisplatin while approximately a 1 : 2 ratio of S- versus N-coordination is observed by transplatin, to be possibly related to the trans effect at the platinum center.

Serine O-sulfation probed by IRMPD spectroscopy.

The sulfation of amino acids is a frequent post-translational modification. It is highly labile, though, and characterizing it by mass spectrometry, an otherwise powerful and widely exploited tool in analytical proteomics, is a challenge. The presently reported study is aimed at revealing the O-sulfation of l-serine and elucidating the effects of protonation and deprotonation on the structure and stability of the ensuing ionic species, [sSer + H](+) and [sSer - H](-). These ions are obtained as gaseous, isolated species by electrospray ionization, trapped in a Paul ion-trap, and sampled by IR multiple photon dissociation (IRMPD) spectroscopy in either the 750-1900 cm(-1) fingerprint range, or the 2900 and 3700 cm(-1) range encompassing the N-H and O-H stretching modes. The recorded IRMPD spectra present diagnostic signatures of the sulfate modification which are missing in the spectra of the native serine ions, [Ser + H](+) and [Ser - H](-). The experimental IRMPD features have been interpreted by comparison with the linear IR spectra of the lowest energy structures that are likely candidates for the sampled ions, calculated at the M06-2X/6-311+G(d,p) level of theory. Evidence is gathered that the most stable conformations of [sSer + H](+) are stabilized by hydrogen bonding interactions between the protonated amino group and both the carbonyl and sulfate oxygens. [sSer - H](-) ions possess a negatively charged sulfate group involved in either a S=O···HN or a S=O···HO hydrogen bond. The experimental IRMPD spectra are consistent with the presence of multiple low-lying structures in a thermally equilibrated population of several species particularly in the case of [sSer - H](-) ions, where the high structural flexibility combined with the presence of a negative charge favors the co-existence of several different H-bonding motifs.

Selective Acetamidine-Based Nitric Oxide Synthase Inhibitors: Synthesis, Docking, and Biological Studies.

N-[(3-Aminomethyl)benzyl]acetamidine derivatives were synthesized and in vitro evaluated as inhibitors of the inducible isoform of nitric oxide synthase (iNOS). Because of the high potency of action and the excellent selectivity over the endothelial nitric oxide synthase (eNOS), compound 10 was ex vivo evaluated on isolated and perfused resistance arteries. The results confirm that compound 10 selectively inhibits the iNOS, without affecting the endothelial isoform. The outcome of the docking studies showed that the hydrophobic interaction is the driving force of the binding process, especially for iNOS, where the binding pocket is characterized by a significant lipophilic region.

Investigation of the molecular similarity in closely related protein systems: The PrP case study.

The amyloid conversion is a massive detrimental modification affecting several proteins upon specific physical or chemical stimuli characterizing a plethora of diseases. In many cases, the amyloidogenic stimuli induce specific structural features to the protein conferring the propensity to misfold and form amyloid deposits. The investigation of mutants, structurally similar to their native isoform but inherently prone to amyloid conversion, may be a viable strategy to elucidate the structural features connected with amyloidogenesis. In this article, we present a computational protocol based on the combination of molecular dynamics (MD) and grid-based approaches suited for the pairwise comparison of closely related protein structures. This method was applied on the cellular prion protein (PrP(C)) as a case study and, in particular, addressed to the quali/quantification of the structural features conferred by either E200K mutations and treatment with CaCl(2), both able to induce the scrapie conversion of PrP. Several schemes of comparison were developed and applied to this case study, and made up suitable of application to other protein systems. At this purpose an in-house python codes has been implemented that, together with the parallelization of the GRID force fields program, will spread the applicability of the proposed computational procedure.