Not All Binding Sites Are Equal: Site Determination and Folding State Analysis of Gas-Phase Protein-Metallodrug Adducts.

Modern approaches in metallodrug research focus on compounds that bind protein targets rather than DNA. However, the identification of protein targets and binding sites is challenging. Using intact mass spectrometry and proteomics, we investigated the binding of the antimetastatic agent RAPTA-C to the model proteins ubiquitin, cytochrome c, lysozyme, and myoglobin. Binding to cytochrome c and lysozyme was negligible. However, ubiquitin bound up to three Ru moieties, two of which were localized at Met1 and His68 as [Ru(cym)], and [Ru(cym)] or [Ru(cym)(PTA)] adducts, respectively. Myoglobin bound up to four [Ru(cym)(PTA)] moieties and five sites were identified at His24, His36, His64, His81/82 and His113. Collision-induced unfolding (CIU) studies via ion-mobility mass spectrometry allowed measuring protein folding as a function of collisional activation. CIU of protein-RAPTA-C adducts showed binding of [Ru(cym)] to Met1 caused a significant compaction of ubiquitin, likely from N-terminal S-Ru-N chelation, while binding of [Ru(cym)(PTA)] to His residues of ubiquitin or myoglobin induced a smaller effect. Interestingly, the folded state of ubiquitin formed by His functionalization was more stable than Met1 metalation. The data suggests that selective metalation of amino acids at different positions on the protein impacts the conformation and potentially the biological activity of anticancer compounds.

AdductHunter: identifying protein-metal complex adducts in mass spectra.

Mass spectrometry (MS) is an analytical technique for molecule identification that can be used for investigating protein-metal complex interactions. Once the MS data is collected, the mass spectra are usually interpreted manually to identify the adducts formed as a result of the interactions between proteins and metal-based species. However, with increasing resolution, dataset size, and species complexity, the time required to identify adducts and the error-prone nature of manual assignment have become limiting factors in MS analysis. AdductHunter is a open-source web-based analysis tool that  automates the peak identification process using constraint integer optimization to find feasible combinations of protein and fragments, and dynamic time warping to calculate the dissimilarity between the theoretical isotope pattern of a species and its experimental isotope peak distribution. Empirical evaluation on a collection of 22 unique MS datasetsshows fast and accurate identification of protein-metal complex adducts in deconvoluted mass spectra.

Discovery, Crystallographic Studies, and Mechanistic Investigations of Novel Phenylalanine Derivatives Bearing a Quinazolin-4-one Scaffold as Potent HIV Capsid Modulators.

Optimization of compound 11L led to the identification of novel HIV capsid modulators, quinazolin-4-one-bearing phenylalanine derivatives, displaying potent antiviral activities against both HIV-1 and HIV-2. Notably, derivatives 12a2 and 21a2 showed significant improvements, with 2.5-fold over 11L and 7.3-fold over PF74 for HIV-1, and approximately 40-fold over PF74 for HIV-2. The X-ray co-crystal structures confirmed the multiple pocket occupation of 12a2 and 21a2 in the binding site. Mechanistic studies revealed a dual-stage inhibition profile, where the compounds disrupted capsid-host factor interactions at the early stage and promoted capsid misassembly at the late stage. Remarkably, 12a2 and 21a2 significantly promoted capsid misassembly, outperforming 11L, PF74, and LEN. The substitution of easily metabolized amide bond with quinolin-4-one marginally enhanced the stability of 12a2 in human liver microsomes compared to controls. Overall, 12a2 and 21a2 highlight their potential as potent HIV capsid modulators, paving the way for future advancements in anti-HIV drug design.

The Leptospermum scoparium (Manuka)-Specific Nectar and Honey Compound 3,6,7-Trimethyllumazine (LepteridineTM) That Inhibits Matrix Metalloproteinase 9 (MMP-9) Activity.

3,6,7-trimethyllumazine (Lepteridine™) is a newly discovered natural pteridine derivative unique to Manuka (Leptospermum scoparium) nectar and honey, with no previously reported biological activity. Pteridine derivative-based medicines, such as methotrexate, are used to treat auto-immune and inflammatory diseases, and Manuka honey reportedly possesses anti-inflammatory properties and is used topically as a wound dressing. MMP-9 is a potential candidate protein target as it is upregulated in recalcitrant wounds and intestinal inflammation. Using gelatin zymography, 40 µg/mL LepteridineTM inhibited the gelatinase activities of both pro- (22%, p < 0.0001) and activated (59%, p < 0.01) MMP-9 forms. By comparison, LepteridineTM exerted modest (~10%) inhibition against a chromogenic peptide substrate and no effect against a fluorogenic peptide substrate. These findings suggest that LepteridineTM may not interact within the catalytic domain of MMP-9 and exerts a negligible effect on the active site hydrolysis of small soluble peptide substrates. Instead, the findings implicate fibronectin II domain interactions by LepteridineTM which impair gelatinase activity, possibly through perturbed tethering of MMP-9 to the gelatin matrix. Molecular modelling analyses were equivocal over interactions at the S1' pocket versus the fibronectin II domain, while molecular dynamic calculations indicated rapid exchange kinetics. No significant degradation of synthetic or natural LepteridineTM in Manuka honey occurred during simulated gastrointestinal digestion. MMP-9 regulates skin and gastrointestinal inflammatory responses and extracellular matrix remodelling. These results potentially implicate LepteridineTM bioactivity in Manuka honey's reported beneficial effects on wound healing via topical application and anti-inflammatory actions in gastrointestinal disorder models via oral consumption.

NlpC/P60 peptidoglycan hydrolases of Trichomonas vaginalis have complementary activities that empower the protozoan to control host-protective lactobacilli.

Trichomonas vaginalis is a human protozoan parasite that causes trichomoniasis, a prevalent sexually transmitted infection. Trichomoniasis is accompanied by a shift to a dysbiotic vaginal microbiome that is depleted of lactobacilli. Studies on co-cultures have shown that vaginal bacteria in eubiosis (e.g. Lactobacillus gasseri) have antagonistic effects on T. vaginalis pathogenesis, suggesting that the parasite might benefit from shaping the microbiome to dysbiosis (e.g. Gardnerella vaginalis among other anaerobes). We have recently shown that T. vaginalis has acquired NlpC/P60 genes from bacteria, expanding them to a repertoire of nine TvNlpC genes in two distinct clans, and that TvNlpCs of clan A are active against bacterial peptidoglycan. Here, we expand this characterization to TvNlpCs of clan B. In this study, we show that the clan organisation of NlpC/P60 genes is a feature of other species of Trichomonas, and that Histomonas meleagridis has sequences related to one clan. We characterized the 3D structure of TvNlpC_B3 alone and with the inhibitor E64 bound, probing the active site of these enzymes for the first time. Lastly, we demonstrated that TvNlpC_B3 and TvNlpC_B5 have complementary activities with the previously described TvNlpCs of clan A and that exogenous expression of these enzymes empower this mucosal parasite to take over populations of vaginal lactobacilli in mixed cultures. TvNlpC_B3 helps control populations of L. gasseri, but not of G. vaginalis, which action is partially inhibited by E64. This study is one of the first to show how enzymes produced by a mucosal protozoan parasite may contribute to a shift on the status of a microbiome, helping explain the link between trichomoniasis and vaginal dysbiosis. Further understanding of this process might have significant implications for treatments in the future.

Platinum(terpyridine) complexes with N-heterocyclic carbene co-ligands: high antiproliferative activity and low toxicity in vivo.

Pt(terpyridine) complexes are well-known DNA intercalators. The introduction of an NHC co-ligand rendered such a complex highly antiproliferative in cancer cells compared to its chlorido derivative. Despite the high potency, zebrafish embryos tolerated the compound well, especially compared to cisplatin. DNA interaction studies support a mode of action related to intercalation.

MtING2 encodes an ING domain PHD finger protein which affects Medicago growth, flowering, global patterns of H3K4me3, and gene expression.

Flowering of the reference legume Medicago truncatula is promoted by winter cold (vernalization) followed by long-day photoperiods (VLD) similar to winter annual Arabidopsis. However, Medicago lacks FLC and CO, key regulators of Arabidopsis VLD flowering. Most plants have two INHIBITOR OF GROWTH (ING) genes (ING1 and ING2), encoding proteins with an ING domain with two anti-parallel alpha-helices and a plant homeodomain (PHD) finger, but their genetic role has not been previously described. In Medicago, Mting1 gene-edited mutants developed and flowered normally, but an Mting2-1 Tnt1 insertion mutant and gene-edited Mting2 mutants had developmental abnormalities including delayed flowering particularly in VLD, compact architecture, abnormal leaves with extra leaflets but no trichomes, and smaller seeds and barrels. Mting2 mutants had reduced expression of activators of flowering, including the FT-like gene MtFTa1, and increased expression of the candidate repressor MtTFL1c, consistent with the delayed flowering of the mutant. MtING2 overexpression complemented Mting2-1, but did not accelerate flowering in wild type. The MtING2 PHD finger bound H3K4me2/3 peptides weakly in vitro, but analysis of gene-edited mutants indicated that it was dispensable to MtING2 function in wild-type plants. RNA sequencing experiments indicated that >7000 genes are mis-expressed in the Mting2-1 mutant, consistent with its strong mutant phenotypes. Interestingly, ChIP-seq analysis identified >5000 novel H3K4me3 locations in the genome of Mting2-1 mutants compared to wild type R108. Overall, our mutant study has uncovered an important physiological role of a plant ING2 gene in development, flowering, and gene expression, which likely involves an epigenetic mechanism.

Mapping the interaction between Trim28 and the KRAB domain at the center of Trim28 silencing of endogenous retroviruses.

Transcription of endogenous retroviral elements are tightly regulated during development by members of the KRAB-containing zinc finger proteins (KRAB-ZFPs) and the co-repressor Trim28 (also known as Kap-1 or Tif1ß). KRAB-ZFPs form the largest family of transcription regulators in mammals and initiate transcriptional silencing by tethering Trim28 to a target locus. Subsequently, Trim28 recruits chromatin modifying effectors resulting in the formation of heterochromatin. In the present study, we identify surface exposed residues on the central six turns of the Trim28 coiled-coil region forming the binding interface for the KRAB domain. Using AlphaFold2 (AF2) we provide high confidence models of the interface between Trim28 and the KRAB domain and identified leucine 301 on each chain of the Trim28 monomer to act as a pin extending into a hydrophobic pocket on the KRAB domain surface. Site directed mutations in the Trim28-KRAB binding interface abolished binding to the KRAB domain. Our work provides a detailed understanding of the specific interactions between the KRAB domain and the Trim28 coiled-coil and how this interaction may be regulated during silencing events.

Anthracenyl Functionalization of Half-Sandwich Carbene Complexes: In Vitro Anticancer Activity and Reactions with Biomolecules.

N-Heterocyclic carbene (NHC) ligands are widely investigated in medicinal inorganic chemistry. Here, we report the preparation and characterization of a series of half-sandwich [M(L)(NHC)Cl2] (M = Ru, Os, Rh, Ir; L = cym/Cp*) complexes with a N-flanking anthracenyl moiety attached to imidazole- and benzimidazole-derived NHC ligands. The anticancer activity of the complexes was investigated in cell culture studies where, in comparison to a Rh derivative with an all-carbon-donor-atom-based ligand (5a), they were found to be cytotoxic with IC50 values in the low micromolar range. The Ru derivative 1a was chosen as a representative for stability studies as well as for biomolecule interaction experiments. It underwent partial chlorido/aqua ligand exchange in DMSO-d6/D2O to rapidly form an equilibrium in aqueous media. The reactions of 1a with biomolecules proceeded quickly and resulted in the formation of adducts with amino acids, DNA, and protein. Hen egg white lysozyme crystals were soaked with 1a, and the crystallographic analysis revealed an interaction with an l-aspartic acid residue (Asp119), resulting in the cleavage of the p-cymene ligand but the retention of the NHC moiety. Cell morphology studies for the Rh analog 3a suggested that the cytotoxicity is exerted via mechanisms different from that of cisplatin.

Structural Analysis of the Menangle Virus P Protein Reveals a Soft Boundary between Ordered and Disordered Regions.

The paramyxoviral phosphoprotein (P protein) is the non-catalytic subunit of the viral RNA polymerase, and coordinates many of the molecular interactions required for RNA synthesis. All paramyxoviral P proteins oligomerize via a centrally located coiled-coil that is connected to a downstream binding domain by a dynamic linker. The C-terminal region of the P protein coordinates interactions between the catalytic subunit of the polymerase, and the viral nucleocapsid housing the genomic RNA. The inherent flexibility of the linker is believed to facilitate polymerase translocation. Here we report biophysical and structural characterization of the C-terminal region of the P protein from Menangle virus (MenV), a bat-borne paramyxovirus with zoonotic potential. The MenV P protein is tetrameric but can dissociate into dimers at sub-micromolar protein concentrations. The linker is globally disordered and can be modeled effectively as a worm-like chain. However, NMR analysis suggests very weak local preferences for alpha-helical and extended beta conformation exist within the linker. At the interface between the disordered linker and the structured C-terminal binding domain, a gradual disorder-to-order transition occurs, with X-ray crystallographic analysis revealing a dynamic interfacial structure that wraps the surface of the binding domain.

Probing the Paradigm of Promiscuity for N-Heterocyclic Carbene Complexes and their Protein Adduct Formation.

Metal complexes can be considered a "paradigm of promiscuity" when it comes to their interactions with proteins. They often form adducts with a variety of donor atoms in an unselective manner. We have characterized the adducts formed between a series of isostructural N-heterocyclic carbene (NHC) complexes with Ru, Os, Rh, and Ir centers and the model protein hen egg white lysozyme by X-ray crystallography and mass spectrometry. Distinctive behavior for the metal compounds was observed with the more labile Ru and Rh complexes targeting mainly a surface l-histidine moiety through cleavage of p-cymene or NHC co-ligands, respectively. In contrast, the more inert Os and Ir derivatives were detected abundantly in an electronegative binding pocket after undergoing ligand exchange of a chlorido ligand for an amino acid side chain. Computational studies supported the binding profiles and hinted at the role of the protein microenvironment for metal complexes eliciting selectivity for specific binding sites on the protein.

Granulovirus PK-1 kinase activity relies on a side-to-side dimerization mode centered on the regulatory αC helix.

The life cycle of Baculoviridae family insect viruses depends on the viral protein kinase, PK-1, to phosphorylate the regulatory protein, p6.9, to induce baculoviral genome release. Here, we report the crystal structure of Cydia pomenella granulovirus PK-1, which, owing to its likely ancestral origin among host cell AGC kinases, exhibits a eukaryotic protein kinase fold. PK-1 occurs as a rigid dimer, where an antiparallel arrangement of the αC helices at the dimer core stabilizes PK-1 in a closed, active conformation. Dimerization is facilitated by C-lobe:C-lobe and N-lobe:N-lobe interactions between protomers, including the domain-swapping of an N-terminal helix that crowns a contiguous ß-sheet formed by the two N-lobes. PK-1 retains a dimeric conformation in solution, which is crucial for catalytic activity. Our studies raise the prospect that parallel, side-to-side dimeric arrangements that lock kinase domains in a catalytically-active conformation could function more broadly as a regulatory mechanism among eukaryotic protein kinases.

Mustards-Derived Terpyridine-Platinum Complexes as Anticancer Agents: DNA Alkylation vs Coordination.

The development of bifunctional platinum complexes with the ability to interact with DNA via different binding modes is of interest in anticancer metallodrug research. Therefore, we report platinum(II) terpyridine complexes to target DNA by coordination and/or through a tethered alkylating moiety. The platinum complexes were evaluated for their in vitro antiproliferative properties against the human cancer cell lines HCT116 (colorectal), SW480 (colon), NCI-H460 (non-small cell lung), and SiHa (cervix) and generally exhibited potent antiproliferative activity although lower than their respective terpyridine ligands. 1H NMR spectroscopy and/or ESI-MS studies on the aqueous stability and reactivity with various small biomolecules, acting as protein and DNA model compounds, were used to establish potential modes of action for these complexes. These investigations indicated rapid binding of complex PtL3 to the biomolecules through coordination to the Pt center, while PtL4 in addition alkylated 9-ethylguanine. PtL3 was investigated for its reactivity to the model protein hen egg white lysozyme (HEWL) by protein crystallography which allowed identification of the Nδ1 atom of His15 as the binding site.

A Combined Spectroscopic and Protein Crystallography Study Reveals Protein Interactions of RhI(NHC) Complexes at the Molecular Level.

While most Rh-N-heterocyclic carbene (NHC) complexes currently investigated in anticancer research contain a Rh(III) metal center, an increasing amount of research is focusing on the cytotoxic activity and mode of action of square-planar [RhCl(COD)(NHC)] (where COD = 1,5-cyclooctadiene) which contains a Rh(I) center. The enzyme thioredoxin reductase (TrxR) and the protein albumin have been proposed as potential targets, but the molecular processes taking place upon protein interaction remain elusive. Herein, we report the preparation of peptide-conjugated and its nonconjugated parent [RhCl(COD)(NHC)] complexes, an in-depth investigation of both their stability in solution, and a crystallographic study of protein interaction. The organorhodium compounds showed a rapid loss of the COD ligand and slow loss of the NHC ligand in aqueous solution. These ligand exchange reactions were reflected in studies on the interaction with hen egg white lysozyme (HEWL) as a model protein in single-crystal X-ray crystallographic investigations. Upon treatment of HEWL with an amino acid functionalized [RhCl(COD)(NHC)] complex, two distinct rhodium adducts were found initially after 7 d of incubation at His15 and after 4 weeks also at Lys33. In both cases, the COD and chlorido ligands had been substituted with aqua and/or hydroxido ligands. While the histidine (His) adduct also indicated a loss of the NHC ligand, the lysine (Lys) adduct retained the NHC core derived from the amino acid l-histidine. In either case, an octahedral coordination environment of the metal center indicates oxidation to Rh(III). This investigation gives the first insight on the interaction of Rh(I)(NHC) complexes and proteins at the molecular level.

The RING domain of TRIM69 promotes higher-order assembly.

Members of the TRIM protein family have been shown to inhibit a range of viral infections. Recently, TRIM69 was identified as a potent inhibitor of Vesicular stomatitis Indiana virus infection, with its inhibition being dependent upon multimerization. Using SEC-MALLS analysis, it is demonstrated that the assembly of TRIM69 is mediated through the RING domain and not the Bbox domain as has been shown for other TRIM proteins. Using X-ray crystallography, the structure of the TRIM69 RING domain has been determined to a resolution of 2.1 Å, the oligomerization interface has been identified and regions outside the four-helix bundle have been observed to form interactions that are likely to support assembly.

Potent Inhibition of Thioredoxin Reductase by the Rh Derivatives of Anticancer M(arene/Cp*)(NHC)Cl2 Complexes.

Metal complexes provide a versatile platform to develop novel anticancer pharmacophores, and they form stable compounds with N-heterocyclic carbene (NHC) ligands, some of which have been shown to inhibit the cancer-related selenoenzyme thioredoxin reductase (TrxR). To expand a library of isostructural NHC complexes, we report here the preparation of RhIII- and IrIII(Cp*)(NHC)Cl2 (Cp* = η5-pentamethylcyclopentadienyl) compounds and comparison of their properties to the RuII- and OsII(cym) analogues (cym = η6-p-cymene). Like the RuII- and OsII(cym) complexes, the RhIII- and IrIII(Cp*) derivatives exhibit cytotoxic activity with half maximal inhibitory concentration (IC50) values in the low micromolar range against a set of four human cancer cell lines. In studies on the uptake and localization of the compounds in cancer cells by X-ray fluorescence microscopy, the Ru and Os derivatives were shown to accumulate in the cytoplasmic region of treated cells. In an attempt to tie the localization of the compounds to the inhibition of the tentative target TrxR, it was surprisingly found that only the Rh complexes showed significant inhibitory activity at IC50 values of ∼1 µM, independent of the substituents on the NHC ligand. This indicates that, although TrxR may be a potential target for anticancer metal complexes, it is unlikely the main target or the sole target for the Ru, Os, and Ir compounds described here, and other targets should be considered. In contrast, Rh(Cp*)(NHC)Cl2 complexes may be a scaffold for the development of TrxR inhibitors.

Structural basis for Fullerene geometry in a human endogenous retrovirus capsid.

The HML2 (HERV-K) group constitutes the most recently acquired family of human endogenous retroviruses, with many proviruses less than one million years old. Many maintain intact open reading frames and provirus expression together with HML2 particle formation are observed in early stage human embryo development and are associated with pluripotency as well as inflammatory disease, cancers and HIV-1 infection. Here, we reconstruct the core structural protein (CA) of an HML2 retrovirus, assemble particles in vitro and employ single particle cryogenic electron microscopy (cryo-EM) to determine structures of four classes of CA Fullerene shell assemblies. These icosahedral and capsular assemblies reveal at high-resolution the molecular interactions that allow CA to form both pentamers and hexamers and show how invariant pentamers and structurally plastic hexamers associate to form the unique polyhedral structures found in retroviral cores.

Gel electrophoresis in combination with laser ablation-inductively coupled plasma mass spectrometry to quantify the interaction of cisplatin with human serum albumin.

Cisplatin and its second and third generation analogues are widely used in the treatment of cancer. To study their reactions with proteins, we present a method based on SDS-PAGE separation and laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) for platinum detection in the reaction between human serum albumin (HSA) and cisplatin. We developed matrix-matched standards of HSA/cisplatin mixtures and used them to quantify the amount of adducts formed at different HSA:cisplatin ratios. We noted that cisplatin incubation with HSA resulted in the formation of higher order HSA n-mers, depending on the amount of cisplatin added. This caused a depletion of the HSA dimer bands, while the majority of HSA was present as the monomer. Inducing the formation of such higher molecular weight species may have an impact on the mode of action of metallodrugs.

A Dissection of Oligomerization by the TRIM28 Tripartite Motif and the Interaction with Members of the Krab-ZFP Family.

TRIM28 (also known as KAP1 or TIF1ß) is the universal co-repressor of the Krüppel-associated box-containing zinc finger proteins (Krab-ZFPs), the largest family of transcription factors in mammals. During early embryogenesis, TRIM28 mediates the transcriptional silencing of many endogenous retroviral elements and genomic imprinted sites. Silencing is initiated by the recruitment of TRIM28 to a target locus by members of the Krab-ZFP. Subsequently, TRIM28 functions as a scaffold protein to recruit chromatin modifying effectors featuring SETDB1, HP1 and the NuRD complex. Although many protein partners involved in silencing have been identified, the molecular basis of the protein interactions that mediate silencing remains largely unclear. In the present study, we identified the first Bbox domain (T28_B1 135-203) as a molecular interface responsible for the formation of higher-order oligomers of TRIM28. The structure of this domain reveals a new interface on the surface of the Bbox domain. Mutants disrupting the interface disrupt the formation of oligomers but have no observed effect on transcriptional silencing defining a single TRIM28 dimer as the functional unit for silencing. Using assembly-deficient mutants, we employed small-angle X-ray scattering and biophysical techniques to characterize binding to member of the Krab-ZFP family. This allows us to narrow and define the binding interface to the center of the coiled-coil region (residues 294-321) of TRIM28 and define mutants that abolish binding to the Krab-ZFP proteins.

Crystal structure of protein tyrosine phosphatase-2 from Cydia pomonella granulovirus.

Many viral genomes encode kinase and phosphatase enzymes to manipulate pathways that are controlled by phosphorylation events. The majority of viral phosphatase genes occur in the Baculoviridae and Poxviridae families of large DNA viruses. The corresponding protein sequences belong to four major homology groups, and structures are currently available for only two of these. Here, the first structure from the third group, the protein tyrosine phosphatase-2 (PTP-2) class of viral phosphatases, is described. It is shown that Cydia pomonella granulovirus PTP-2 has the same general fold and active-site architecture as described previously for other phosphatases, in the absence of significant sequence homology. Additionally, it has a novel C-terminal extension in an area corresponding to the interface of dimeric poxvirus phosphatases belonging to the Tyr-Ser protein phosphatase homology group.