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.

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.

Radiographic considerations for pediatric supracondylar humerus fractures.

Although supracondylar humerus fractures are common pediatric injuries, guidelines for postoperative imaging remain unclear. This study's purpose was to evaluate decision-making at various points in the postoperative period. The secondary objective was to compare the use of mini C arm fluoroscopy and flat plate X-rays at the first postoperative visit. A retrospective, cohort study was performed at one level I trauma center. Patients ages 1 to 14 with extension Gartland type II-IV supracondylar fractures sustained between January 2013 and May 2020 and treated with closed or open reduction and percutaneous fixation were included. Data collected included demographics, fracture characteristics, and imaging information. Of 553 patients who underwent surgery, 375 (67.8%) received intraoperative images after casting; none resulted in an intraoperative intervention. Of 463 patients with imaging at first follow-up, nine (1.9%) had a management modification, including seven for loss of reduction, all determined by the original operating surgeon. The method of imaging, did not differ significantly with respect to revision surgery. Twenty-six (4.0%) of 532 patients with imaging at pin removal received additional casting after pin removal, but no patients had their pins retained. This retrospective study examined the efficacy of imaging in pediatric supracondylar fractures. Intraoperative, postcasting images did not change management and should be discontinued. Imaging at first follow-up can be useful in identifying patients with loss of reduction and mini C arm serves as a viable alternative to standard X-rays. Finally, imaging at pin removal resulted in additional casting only in type III fractures. Level of evidence: Level III-retrospective, cohort study.

Perforation of the Knee Joint Following Antegrade Intramedullary Nailing of a Comminuted Femoral Diaphyseal Fracture: A Case Report.

A 63-year-old man with a comminuted spiral femoral shaft fracture was treated with closed reduction and internal fixation with a cephalomedullary nail. Two weeks postoperatively, one of the two static distal interlocking bolts began backing out and was removed. The nail ultimately migrated distally and perforated the knee joint at four months postoperatively. The patient was successfully treated with an exchange nail and percutaneous bone graft to the fracture site. A single static distal interlocking bolt may be inadequate to maintain length in a healing comminuted spiral femur shaft. Multiple distal interlocking bolts should be in place until the completion of fracture healing.

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.

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.

Triazolyl-Functionalized N-Heterocyclic Carbene Half-Sandwich Compounds: Coordination Mode, Reactivity and in†vitro Anticancer Activity.

We report investigations on the anticancer activity of organometallic [MII/III (η6 -p-cymene/η5 -pentamethylcyclopentadienyl)] (M=Ru, Os, Rh, and Ir) complexes of N-heterocyclic carbenes (NHCs) substituted with a triazolyl moiety. Depending on the precursors, the NHC ligands displayed either mono- or bidentate coordination via the NHC carbon atom or as N,C-donors. The metal complexes were investigated for their stability in aqueous solution, with the interpretation supported by density functional theory calculations, and reactivity to biomolecules. In vitro cytotoxicity studies suggested that the nature of both the metal center and the lipophilicity of the ligand determine the biological properties of this class of compounds. The IrIII complex 5 d bearing a benzimidazole-derived ligand was the most cytotoxic with an IC50 value of 10 µM against NCI-H460 non-small cell lung carcinoma cells. Cell uptake and distribution studies using X-ray fluorescence microscopy revealed localization of 5 d in the cytoplasm of cancer cells.

Three-dimensional Computed Tomography Posterior Iliac Oblique Images Enhance Preoperative Planning for Acetabular Fracture Surgery.

The advent of computed tomography and development of three-dimensional (3-D) reconstructions has allowed for profound advances in the understanding of complex acetabular fractures. The authors sought to determine the impact of 3-D reconstructions on understanding of the morphology of these injuries. A survey of 20 fellowship-trained orthopaedic trauma surgeons was undertaken to assess the utility of these reconstructions on understanding three complex posterior acetabulum fractures. Respondents noted significantly better understanding of posterior wall and transverse-posterior wall fracture patterns compared to a posterior column-posterior wall pattern when utilizing two-dimensional imaging only. The respondent#x02019;s understanding of all three patterns was improved with the addition of 3-D reconstructions. With regards to individual images, posterior iliac oblique reconstructions obtained at 36-degree and 54-degree from posterior were reported to be most helpful in improving understanding of fracture morphology. Three-dimensional reconstructions of posterior acetabular fractures are effective in enhancing understanding of fracture morphology. (Journal of Surgical Orthopaedic Advances 30(1):050-054, 2021).

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.

Anatomic considerations for retrograde fibular medullary screw insertion: a cadaveric study.

OBJECTIVES: A retrograde fibular medullary screw may be utilized in certain fractures about the ankle. The purpose of this study is to investigate the anatomic considerations of a retrograde medullary screw inserted from a lateral starting point to nearby anatomic structures about the distal fibula. METHODS: Ten fresh-frozen cadaveric lower extremities were utilized. A 1.6-mm Kirschner wire was inserted into the distal fibula from a far-lateral starting point. A 3.2-mm cannulated drill bit was then inserted over the Kirschner wire. After placement of the drill bit, dissection of the lateral ankle was undertaken. The proximity of nearby anatomic structures to the drill bit was measured using calipers. A 4.5-mm cortical screw was then inserted using fluoroscopic guidance. Measurements were then taken again to assess the relationship of the screw head to adjacent structures. RESULTS: Mean distance from drill bit to nearby structures is as follows: Peroneus longus tendon 4.56 mm, peroneus brevis tendon 6.62 mm, sural nerve 4.13 mm, superior peroneal retinaculum 7.52 mm, inferior peroneal retinaculum 6.61 mm, anterior talofibular ligament (ATFL) 6.1 mm, calcaneofibular ligament (CFL) 6.7 mm. Average distance from 4.5-mm screw head to nearby structures is as follows: peroneus longus tendon 6.79 mm, peroneus brevis tendon 6.73 mm, ATFL 4.16 mm, CFL 5.14 mm, lateral talar process 9.41 mm. CONCLUSION: Retrograde medullary fibular screw fixation may be safely carried out through a lateral start point. Anatomic structures about the lateral ankle are nearby but not immediately adjacent to the drill bit.

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.

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.

A Comparison of Complications and Union Rates in Intramedullary Nailing of Femoral Shaft Fractures Treated With Open Versus Closed Reduction.

Intramedullary rod fixation is a common technique for treatment of femoral shaft fractures, with both open and closed reduction techniques described. The purpose of this study was to assess union and complication rates among patients treated with open vs closed reduction and intramedullary nailing of closed femoral shaft fractures. A total of 107 patients undergoing intramedullary fixation of nonpathologic femoral shaft fractures (AO/OTA type 32) between January 2012 and June 2017 were retrospectively studied. Those undergoing open reduction prior to intramedullary nailing were compared with those undergoing closed reduction. The primary outcome analyzed was union rate. Secondary outcomes were time to union, complications necessitating return to the operating room, and operative times. Mean follow-up was 14 months in both groups (range, 6-48 months). Of the 107 patients, 34.6% (n=37) underwent open reduction and 65.4% (n=70) underwent closed reduction. Patients in the open reduction group had rates of union (89.1%, 33 of 37) similar to those of patients in the closed reduction group (92.9%, 65 of 70; P=.378). Patients in the open reduction group who had union did so in a mean of 6.2 months (range, 3-12 months) vs a mean of 5.4 months (range, 2-11 months) in the closed reduction group (P=.13). Six patients (16.2%) in the open reduction group and 6 patients (8.6%) in the closed reduction group had a postoperative complication requiring return to the operating room (P=.18). Open reduction and intramedullary nailing results in rates of union, time to union, and rates of significant complications similar to those of closed reduction and intramedullary nailing. [Orthopedics. 2020; 43(2): 103-107.].

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.

Correction: Unexpected arene ligand exchange results in the oxidation of an organoruthenium anticancer agent: the first X-ray structure of a protein-Ru(carbene) adduct.

Correction for 'Unexpected arene ligand exchange results in the oxidation of an organoruthenium anticancer agent: the first X-ray structure of a protein-Ru(carbene) adduct' by Matthew P. Sullivan et al., Chem. Commun., 2018, 54, 6120-6123.

A method for analyzing the composition of viral nucleoprotein complexes, produced by heterologous expression in bacteria.

Viral genomes are protected and organized by virally encoded packaging proteins. Heterologous production of these proteins often results in formation of particles resembling the authentic viral capsid or nucleocapsid, with cellular nucleic acids packaged in place of the viral genome. Quantifying the total protein and nucleic acid content of particle preparations is a recurrent biochemical problem. We describe a method for resolving this problem, developed when characterizing particles resembling the Menangle Virus nucleocapsid. The protein content was quantified using the biuret assay, which is largely independent of amino acid composition. Bound nucleic acids were quantified by determining the phosphorus content, using inductively coupled plasma mass spectrometry. Estimates for the amount of RNA packaged within the particles were consistent with the structurally-characterized packaging mechanism. For a bacterially-produced nucleoprotein complex, phosphorus usually provides a unique elemental marker of bound nucleic acids, hence this method of analysis should be routinely applicable.

Unexpected arene ligand exchange results in the oxidation of an organoruthenium anticancer agent: the first X-ray structure of a protein-Ru(carbene) adduct.

The first X-ray structures of adducts formed between a RuII(N-heterocyclic carbene)(η6-p-cymene) compound and a protein are reported. Coordination to the protein induced the cleavage of the cymene ligand and EPR spectroscopy demonstrated the oxidation of the Ru centre.

Antitumor Metallodrugs that Target Proteins.

Anticancer platinum-based drugs are widely used in the treatment of a variety of tumorigenic diseases. They have been identified to target DNA and thereby induce apoptosis in cancer cells. Their reactivity to biomolecules other than DNA has often been associated with side effects that many cancer patients experience during chemotherapy. The development of metal compounds that target proteins rather than DNA has the potential to overcome or at least reduce the disadvantages of commonly used chemotherapeutics. Many exciting new metal complexes with novel modes of action have been reported and their anticancer activity was linked to selective protein interaction that may lead to improved accumulation in the tumor, higher selectivity and/or enhanced antiproliferative efficacy. The development of new lead structures requires bioanalytical methods to confirm the hypothesized modes of action or identify new, previously unexplored biological targets and pathways. We have selected original developments for review in this chapter and highlighted compounds on track toward clinical application.

Current Management of Talar Fractures.

Talar fractures are some of the most challenging injuries that orthopaedic traumatologists manage. The current knowledge of functional alterations with regard to malreduction of talar fractures is well established. Decision making with regard to timing, approach, and implant selection as well as strategies to help achieve accurate restoration of talar anatomy substantially affect outcomes and must be carefully considered. Perfect anatomic talar reconstruction should always be attempted, and orthopaedic surgeons should have a strong working knowledge of the vascular, three-dimensional, and radiographic anatomy of the talus before performing talar surgery. Almost the entire talus is surgically accessible via several approaches, all of which surgeons should be clinically familiar with to optimize reduction and fixation and safely preserve the soft-tissue envelope. Furthermore, surgeons must appreciate the plantarmedial vascular area of the talus, which must be avoided during dissection. The complication rates in patients who have talar fractures are high, particularly in those who have talar neck and talar body fractures; therefore, patients should be counseled on their expected outcome, with a specific discussion on the risk of osteonecrosis and subtalar arthritis.