Reinforcing Protein Biochemistry: A Two-Week Experiment Studying Iron(III) Binding by the Transferrin Protein through Stoichiometric Determination, Stability Analysis, and Visualization of the Binding Site.

The two-week protein biochemistry experience described herein focuses on reinforcing key biochemical concepts and achieving significant learning domain accomplishments for students (Content Knowledge, Logical Mathematical Reasoning, Visualization, Information Literacy, and Knowledge Integration) and valuable teaching opportunities for instructors. The experience encompasses an exploration of the transport protein serum transferrin as an important regulator of Fe(III) biochemistry and incorporates techniques to assess protein-metal stoichiometry and protein stability and to perform molecular visualization. Students gain practical experience in utilizing spectrophotometric analysis for constructing stoichiometric curves, in performing urea-PAGE, and in applying the PyMOL program to evaluate metal coordination at a protein binding site and the associated protein structural change. The learning and teaching accomplishments provide valuable skills that can be extended into research and translated to other teaching formats.

Exploring the pH dependent aqueous speciation of metal complexes through UV-Vis spectroscopy.

Coordination chemistry is a major component of the undergraduate inorganic chemistry curriculum and yet the presentation of the material can be cumbersome due to the limitations of the course typically being taught in one semester. Also, because of the large scope of this branch of chemistry encompassing all of the elements, the course design has not been standardized. These factors result in some important coordination chemistry themes being given insufficient development. Herein we propose a novel activity to formally introduce metal complex aqueous speciation in a holistic active-learning manner that includes a lecture component and hands-on experience. This topic has real world relevance and contextualizes many important coordination concepts. It would extend student comprehension about the intricate factors that affect metal complexation in an aqueous solution environment by focusing on the influence of pH. The activity explores the pH dependent speciation of the well-characterized interaction between Fe(III) and 2,3-dihydroxynapthalene-6-sulfonate and reveals the colorful changes in species throughout the pH range of 0 to 13. Students learn how to generate speciation plots and to understand the ultraviolet-visible (UV-Vis) electronic absorption spectroscopy of transition metal compounds to be able to analyze the source of color that they observe. Assessment of the activity was conducted with 24 students who completed a Likert scale survey and responded to open-ended questions. The activity was then applied in actual course settings in which student comprehension was quantitatively evaluated. The activity can be easily adapted to students of different stages of academic development from elementary to college students.

Exploring titanium(IV) chemical proximity to iron(III) to elucidate a function for Ti(IV) in the human body.

Despite its natural abundance and widespread use as food, paint additive, and in bone implants, no specific biological function of titanium is known in the human body. High concentrations of Ti(IV) could result in cellular toxicity, however, the absence of Ti toxicity in the blood of patients with titanium bone implants indicates the presence of one or more biological mechanisms to mitigate toxicity. Similar to Fe(III), Ti(IV) in blood binds to the iron transport protein serum transferrin (sTf), which gives credence to the possibility of its cellular uptake mechanism by transferrin-directed endocytosis. However, once inside the cell, how sTf bound Ti(IV) is released into the cytoplasm, utilized, or stored remain largely unknown. To explain the molecular mechanisms involved in Ti use in cells we have drawn parallels with those for Fe(III). Based on its chemical similarities with Fe(III), we compare the biological coordination chemistry of Fe(III) and Ti(IV) and hypothesize that Ti(IV) can bind to similar intracellular biomolecules. The comparable ligand affinity profiles suggest that at high Ti(IV) concentrations, Ti(IV) could compete with Fe(III) to bind to biomolecules and would inhibit Fe bioavailability. At the typical Ti concentrations in the body, Ti might exist as a labile pool of Ti(IV) in cells, similar to Fe. Ti could exhibit different types of properties that would determine its cellular functions. We predict some of these functions to mimic those of Fe in the cell and others to be specific to Ti. Bone and cellular speciation and localization studies hint toward various intracellular targets of Ti like phosphoproteins, DNA, ribonucleotide reductase, and ferritin. However, to decipher the exact mechanisms of how Ti might mediate these roles, development of innovative and more sensitive methods are required to track this difficult to trace metal in vivo.

Expanding the Therapeutic Potential of the Iron Chelator Deferasirox in the Development of Aqueous Stable Ti(IV) Anticancer Complexes.

The recent X-ray structure of titanium(IV)-bound human serum transferrin (STf) exhibiting citrate as a synergistic anion reveals a difference in Ti(IV) coordination versus iron(III), the metal endogenously delivered by the protein to cells. This finding enriches our bioinspired drug design strategy for Ti(IV)-based anticancer therapeutics, which applies a family of Fe(III) chelators termed chemical transferrin mimetic (cTfm) ligands to inhibit Fe bioavailability in cancer cells. Deferasirox, a drug used for iron overload disease, is a cTfm ligand that models STf coordination to Fe(III), favoring Fe(III) binding versus Ti(IV). This metal affinity preference drives deferasirox to facilitate the release of cytotoxic Ti(IV) intracellularly in exchange for Fe(III). An aqueous speciation study performed by potentiometric titration from pH 4 to 8 with micromolar levels of Ti(IV) deferasirox at a 1:2 ratio reveals exclusively Ti(deferasirox)2 in solution. The predominant complex at pH 7.4, [Ti(deferasirox)2]2-, exhibits the one of the highest aqueous stabilities observed for a potent cytotoxic Ti(IV) species, demonstrating little dissociation even after 1 month in cell culture media. UV-vis and 1H NMR studies show that the stability is unaffected by the presence of biomolecular Ti(IV) binders such as citrate, STf, and albumin, which have been shown to induce dissociation or regulate cellular uptake and can alter the activity of other antiproliferative Ti(IV) complexes. Kinetic studies on [Ti(deferasirox)2]2- transmetalation with Fe(III) show that a labile Fe(III) source is required to induce this process. The initial step of this process occurs on the time scale of minutes, and equilibrium for the complete transmetalation is reached on a time scale of hours to a day. This work reveals a mechanism to deliver Ti(IV) compounds into cells and trigger Ti(IV) release by a labile Fe(III) species. Cellular studies including other cTfm ligands confirm the Fe(III) depletion mechanism of these compounds and show their ability to induce early and late apoptosis.

Unusual Synergism of Transferrin and Citrate in the Regulation of Ti(IV) Speciation, Transport, and Toxicity.

Human serum transferrin (sTf) is a protein that mediates the transport of iron from blood to cells. Assisted by the synergistic anion carbonate, sTf transports Fe(III) by binding the metal ion in a closed conformation. Previous studies suggest sTf's role as a potential transporter of other metals such as titanium. Ti is a widely used metal in colorants, foods, and implants. A substantial amount of Ti is leached into blood from these implants. However, the fate of the leached Ti and its transport into the cells is not known. Understanding Ti interaction with sTf assumes a greater significance with our ever increasing exposure to Ti in the form of implants. On the basis of in vitro studies, it was speculated that transferrin can bind Ti(IV) assisted by a synergistic anion. However, the role and identity of the synergistic anion(s) and the conformational state in which sTf binds Ti(IV) are not known. Here we have solved the first X-ray crystal structure of a Ti(IV)-bound sTf. We find that sTf binds Ti(IV) in an open conformation with both carbonate and citrate as synergistic anions at the metal binding sites, an unprecedented role for citrate. Studies with cell lines suggest that Ti(IV)-sTf is transported into cells and that sTf and citrate regulate the metal's blood speciation and attenuate its cytotoxic property. Our results provide the first glimpse into the citrate-transferrin synergism in the regulation of Ti(IV) bioactivity and offers insight into the future design of Ti(IV)-based anticancer drugs.

Combining Stimulus-Triggered Release and Active Targeting Strategies Improves Cytotoxicity of Cytochrome c Nanoparticles in Tumor Cells.

Proteins often possess highly specific biological activities that make them potential therapeutics, but their physical and chemical instabilities during formulation, storage, and delivery have limited their medical use. Therefore, engineering of nanosized vehicles to stabilize protein therapeutics and to allow for targeted treatment of complex diseases, such as cancer, is of considerable interest. A micelle-like nanoparticle (NP) was designed for both, tumor targeting and stimulus-triggered release of the apoptotic protein cytochrome c (Cyt c). This system is composed of a Cyt c NP stabilized by a folate-receptor targeting amphiphilic copolymer (FA-PEG-PLGA) attached to Cyt c through a redox-sensitive bond. FA-PEG-PLGA-S-S-Cyt c NPs exhibited excellent stability under extracellular physiological conditions, whereas once in the intracellular reducing environment, Cyt c was released from the conjugate. Under the same conditions, the folate-decorated NP reduced folate receptor positive HeLa cell viability to 20%, while the same complex without FA only reduced it to 80%. Confocal microscopy showed that the FA-PEG-PLGA-S-S-Cyt c NPs were internalized by HeLa cells and were capable of endosomal escape. The specificity of the folate receptor-mediated internalization was confirmed by the lack of uptake by two folate receptor deficient cell lines: A549 and NIH-3T3. Finally, the potential as antitumor therapy of our folate-decorated Cyt c-based NPs was confirmed with an in vivo brain tumor model. In conclusion, we were able to create a stable, selective, and smart nanosized Cyt c delivery system.

Purification and characterization of a cytochrome c with novel caspase-3 activation activity from the pathogenic fungus Rhizopus arrhizus.

BACKGROUND: Members of Rhizopus species are the most common cause of mucormycosis, a rare but often fatal fungal infection. Host induced pathogen apoptosis and pathogen induced host cell apoptosis are often involved in fungal infections. In many organisms, the release of mitochondrial cytochrome c can trigger apoptosis by activating caspase proteases, but the role of fungal cytochrome c in apoptosis remains unknown. RESULTS: DNA sequence encoding Rhizopus arrhizus cytochrome c was cloned and expressed in E. coli. Both native and recombinant cytochrome c were purified using ion exchange followed by gel filtration chromatography. The identities of purified proteins were confirmed by MALDI-MS and UV-Visible spectroscopy. For the first time, we demonstrated that Rhizopus arrhizus cytochrome c could activate human capspase-3 in HeLa cell extracts. We also found that Rhizopus arrhizus cytochrome c has redox potential, peroxidase activity, and spectral properties similar to human and horse cytochrome c proteins. CONCLUSIONS: Rhizopus arrhizus cytochrome c can activate human caspase-3 in HeLa cell extracts and it possesses similar physical and spectral properties as human and horse cytochrome c. This protein was found to have a previously unknown potential to activate human caspase-3, an important step in the apoptosis cascade.

Cytotoxicity of a Ti(IV) compound is independent of serum proteins.

Titanium(IV) compounds are excellent anticancer drug candidates, but they have yet to find success in clinical applications. A major limitation in developing further compounds has been a general lack of understanding of the mechanism governing their bioactivity. To determine factors necessary for bioactivity, we tested the cytotoxicity of different ligand compounds in conjunction with speciation studies and mass spectrometry bioavailability measurements. These studies demonstrated that the Ti(IV) compound of N,N'-di(o-hydroxybenzyl)ethylenediamine-N,N'-diacetic acid (HBED) is cytotoxic to A549 lung cancer cells, unlike those of citrate and naphthalene-2,3-diolate. Although serum proteins are implicated in the activity of Ti(IV) compounds, we found that these interactions do not play a role in [TiO(HBED)](-) activity. Subsequent compound characterization revealed ligand properties necessary for activity. These findings establish the importance of the ligand in the bioactivity of Ti(IV) compounds, provides insights for developing next-generation Ti(IV) anticancer compounds, and reveal [TiO(HBED)](-) as a unique candidate anticancer compound.

Applying the Fe(III) binding property of a chemical transferrin mimetic to Ti(IV) anticancer drug design.

As an endogenous serum protein binder of Ti(IV), transferrin (Tf) serves as an excellent vehicle to stabilize the hydrolysis prone metal ion and successfully transport it into cells. This transporting role is thought to be central to Ti(IV)'s anticancer function, but efforts to synthesize Ti(IV) compounds targeting transferrin have not produced a drug. Nonetheless, the Ti(IV) transferrin complex (Ti2Tf) greatly informs on a new Ti(IV)-based anticancer drug design strategy. Ti2Tf interferes with cellular uptake of Fe(III), which is particularly detrimental to cancer cells because of their higher requirement for iron. Ti(IV) compounds of chemical transferrin mimetic (cTfm) ligands were designed to facilitate Ti(IV) activity by attenuating Fe(III) intracellular levels. In having a higher affinity for Fe(III) than Ti(IV), these ligands feature the appropriate balance between stability and lability to effectively transport Ti(IV) into cancer cells, release Ti(IV) via displacement by Fe(III), and deplete the intracellular Fe(III) levels. The cTfm ligand N,N'-di(o-hydroxybenzyl)ethylenediamine-N,N'-diacetic acid (HBED) was selected to explore the feasibility of the design strategy. Kinetic studies on the Fe(III) displacement process revealed that Ti(IV) can be transported and released into cells by HBED on a physiologically relevant time scale. Cell viability studies using A549 cancerous and MRC5 normal human lung cells and testing the cytotoxicity of HBED and its Ti(IV), Fe(III), and Ga(III) compounds demonstrate the importance of Fe(III) depletion in the proposed drug design strategy and the specificity of the strategy for Ti(IV) activity. The readily derivatized cTfm ligands demonstrate great promise for improved Ti(IV) anticancer drugs.

Thin-Film Organic Heteroepitaxy.

Incorporating crystalline organic semiconductors into electronic devices requires understanding of heteroepitaxy given the ubiquity of heterojunctions in these devices. However, while rules for commensurate epitaxy of covalent or ionic inorganic material systems are known to be dictated by lattice matching constraints, rules for heteroepitaxy of molecular systems are still being written. Here, it is found that lattice matching alone is insufficient to achieve heteroepitaxy in molecular systems, owing to weak intermolecular forces that describe molecular crystals. It is found that, in addition, the lattice matched plane also must be the lowest energy surface of the adcrystal to achieve one-to-one commensurate molecular heteroepitaxy over a large area. Ultraviolet photoelectron spectroscopy demonstrates the lattice matched interface to be of higher electronic quality than a disordered interface of the same materials.

Synthesis of Novel Heterocyclic Ferrocenyl Chalcones and Their Biological Evaluation.

Breast cancer is currently the most commonly diagnosed cancer, with 287,850 new cases estimated for 2022 as reported by the American Cancer Society. Therefore, finding an effective treatment for this disease is imperative. Chalcones are α,ß-unsaturated systems found in nature. These compounds have shown a wide array of biological activities, making them popular synthetic targets. Chalcones consist of two aromatic substituents connected by an enone bridge; this arrangement allows for a large number of derivatives. Given the biological relevance of these compounds, novel ferrocene-heterocycle-containing chalcones were synthesized and characterized based on a hybrid drug design approach. These heterocycles included thiophene, pyrimidine, thiazolyl, and indole groups. Fourteen novel heterocyclic ferrocenyl chalcones were synthesized and characterized. Herein, we also report their cytotoxicity against triple-negative breast cancer cell lines MDA-MB-231 and 4T1 and the noncancer lung cell line MRC-5. System 3 ferrocenyl chalcones displayed superior anticancer properties compared to their system 1 analogues. System 3 chalcones bearing five-membered heterocyclic substituents (thiophene, pyrazole, pyrrole, and pyrimidine) were the most active toward the MDA-MB-231 cancer cell line with IC50 values from 6.59 to 12.51 µM. Cytotoxicity of the evaluated compounds in the 4T1 cell line exhibited IC50 values from 13.23 to 213.7 µM. System 3 pyrazole chalcone had consistent toxicity toward both cell lines (IC50 ∼ 13 µM) as well as promising selectivity relative to the noncancer MRC-5 control. Antioxidant activity was also evaluated, where, contrary to anticancer capabilities, system 1 ferrocenyl chalcones were superior to their system 3 analogues. Antioxidant activity comparable to that of ascorbic acid was observed for thiophene-bearing ferrocenyl chalcone with EC50 = 31 µM.

Exploring Titanium(IV) Complexes as Potential Antimicrobial Compounds.

Due to the rapid mutation of pathogenic microorganisms, drug-resistant superbugs have evolved. Antimicrobial-resistant germs may share their resistance genes with other germs, making them untreatable. The search for more combative antibiotic compounds has led researchers to explore metal-based strategies centered on perturbing the bioavailability of essential metals in microbes and examining the therapeutic potential of metal complexes. Given the limited knowledge on the application of titanium(IV), in this work, eight Ti(IV) complexes and some of their corresponding ligands were screened by the Community for Open Antimicrobial Drug Discovery for antimicrobial activity. The compounds were selected for evaluation because of their low cytotoxic/antiproliferative behavior against a human non-cancer cell line. At pH 7.4, these compounds vary in terms of their solution stability and ligand exchange lability; therefore, an assessment of their solution behavior provides some insight regarding the importance of the identity of the metal compound to the antimicrobial therapeutic potential. Only one compound, Ti(deferasirox)2, exhibited promising inhibitory activity against the Gram-positive bacteria methicillin-resistant Staphylococcus aureus and minimal toxicity against human cells. The ability of this compound to undergo transmetalation with labile Fe(III) sources and, as a consequence, inhibit Fe bioavailability and ribonucleotide reductase is evaluated as a possible mechanism for its antibiotic effect.

Investigating endogenous peptides and peptidases using peptidomics.

Rather than simply being protein degradation products, peptides have proven to be important bioactive molecules. Bioactive peptides act as hormones, neurotransmitters, and antimicrobial agents in vivo. The dysregulation of bioactive peptide signaling is also known to be involved in disease, and targeting peptide hormone pathways has been a successful strategy in the development of novel therapeutics. The importance of bioactive peptides in biology has spurred research to elucidate the function and regulation of these molecules. Classical methods for peptide analysis have relied on targeted immunoassays, but certain scientific questions necessitated a broader and more detailed view of the peptidome--all the peptides in a cell, tissue, or organism. In this review we discuss how peptidomics has emerged to fill this need through the application of advanced liquid chromatography--tandem mass spectrometry (LC-MS/MS) methods that provide unique insights into peptide activity and regulation.

A peptidomics strategy to elucidate the proteolytic pathways that inactivate peptide hormones.

Proteolysis plays a key role in regulating the levels and activity of peptide hormones. Characterization of the proteolytic pathways that cleave peptide hormones is of basic interest and can, in some cases, spur the development of novel therapeutics. The lack, however, of an efficient approach to identify endogenous fragments of peptide hormones has hindered the elucidation of these proteolytic pathways. Here, we apply a mass spectrometry (MS) based peptidomics approach to characterize the intestinal fragments of peptide histidine isoleucine (PHI), a hormone that promotes glucose-stimulated insulin secretion (GSIS). Our approach reveals a proteolytic pathway in the intestine that truncates PHI at its C-terminus to produce a PHI fragment that is inactive in a GSIS assay, a result that provides a potential mechanism of PHI regulation in vivo. Differences between these in vivo peptidomics studies and in vitro lysate experiments, which showed N- and C-terminal processing of PHI, underscore the effectiveness of this approach to discover physiologically relevant proteolytic pathways. Moreover, integrating this peptidomics approach with bioassays (i.e., GSIS) provides a general strategy to reveal proteolytic pathways that may regulate the activity of peptide hormones.

On the evolutionary significance and metal-binding characteristics of a monolobal transferrin from Ciona intestinalis.

Transferrins are a family of proteins that bind and transport Fe(III). Modern transferrins are typically bilobal and are believed to have evolved from an ancient gene duplication of a monolobal form. A novel monolobal transferrin, nicatransferrin (nicaTf), was identified in the primitive ascidian species Ciona intestinalis that possesses the characteristic features of the proposed ancestral Tf protein. In this work, nicaTf was expressed in Pichia pastoris. Extensive solution studies were performed on nicaTf, including UV-vis, fluorescence, CD, EPR and NMR spectroscopies, and electrospray time-of-flight mass spectrometry. The expressed protein is nonglycosylated, unlike the protein isolated from the organism. This property does not affect its ability to bind Fe(III). However, Fe(III)-bound nicaTf displays important spectral differences from other Fe(III)-bound transferrins, which are likely the consequence of differences in metal coordination. Coordination differences could also account for the weaker affinity of nicaTf for Fe(III) (log K = 18.5) compared with bilobal human serum transferrin (HsTf) (log K = 22.5 and 21.4). The Fe-nicaTf complex is not labile, as indicated by slow metal removal kinetics by the high-affinity chelator tiron at pH 7.4. The protein alternatively binds up to one equivalent of Ti(IV) or V(V), which suggests that it may transport nonferric metals. These solution studies provide insight into the structure and function of the primitive monolobal transferrin of C. intestinalis for comparison with higher order bilobal transferrins. They suggest that a major advantage for the evolution of modern transferrins, dominantly of bilobal form, is stronger Fe(III) affinity because of cooperativity.

Iron Chelator Transmetalative Approach to Inhibit Human Ribonucleotide Reductase.

Efforts directed at curtailing the bioavailability of intracellular iron could lead to the development of broad-spectrum anticancer drugs given the metal's role in cancer proliferation and metastasis. Human ribonucleotide reductase (RNR), the key enzyme responsible for synthesizing the building blocks of DNA replication and repair, depends on Fe binding at its R2 subunit to activate the catalytic R1 subunit. This work explores an intracellular iron chelator transmetalative approach to inhibit RNR using the titanium(IV) chemical transferrin mimetic (cTfm) compounds Ti(HBED) and Ti(Deferasirox)2. Whole-cell EPR studies reveal that the compounds can effectively attenuate RNR activity though seemingly causing different changes to the labile iron pool that may account for differences in their potency against cells. Studies of Ti(IV) interactions with the adenosine nucleotide family at pH 7.4 reveal strong metal binding and extensive phosphate hydrolysis, which suggest the capacity of the metal to disturb the nucleotide substrate pool of the RNR enzyme. By decreasing intracellular Fe bioavailability and altering the nucleotide substrate pool, the Ti cTfm compounds could inhibit the activity of the R1 and R2 subunits of RNR. The compounds arrest the cell cycle in the S phase, indicating suppressed DNA replication, and induce apoptotic cell death. Cotreatment cell viability studies with cisplatin and Ti(Deferasirox)2 reveal a promising synergism between the compounds that is likely owed to their distinct but complementary effect on DNA replication.

Cytochrome c: Using Biological Insight toward Engineering an Optimized Anticancer Biodrug.

The heme protein cytochrome c (Cyt c) plays pivotal roles in cellular life and death processes. In the respiratory chain of mitochondria, it serves as an electron transfer protein, contributing to the proliferation of healthy cells. In the cell cytoplasm, it activates intrinsic apoptosis to terminate damaged cells. Insight into these mechanisms and the associated physicochemical properties and biomolecular interactions of Cyt c informs on the anticancer therapeutic potential of the protein, especially in its ability to subvert the current limitations of small molecule-based chemotherapy. In this review, we explore the development of Cyt c as an anticancer drug by identifying cancer types that would be receptive to the cytotoxicity of the protein and factors that can be finetuned to enhance its apoptotic potency. To this end, some information is obtained by characterizing known drugs that operate, in part, by triggering Cyt c induced apoptosis. The application of different smart drug delivery systems is surveyed to highlight important features for maintaining Cyt c stability and activity and improving its specificity for cancer cells and high drug payload release while recognizing the continuing limitations. This work serves to elucidate on the optimization of the strategies to translate Cyt c to the clinical market.

Expanding the dipeptidyl peptidase 4-regulated peptidome via an optimized peptidomics platform.

In recent years, the biological sciences have seen a surge in the development of methods, including high-throughput global methods, for the quantitative measurement of biomolecule levels (i.e., RNA, proteins, metabolites) from cells and tissues. Just as important as quantitation of biomolecules has been the creation of approaches that uncover the regulatory and signaling connections between biomolecules. Our specific interest is in understanding peptide metabolism in a physiological setting, and this has led us to develop a multidisciplinary approach that integrates genetics, analytical chemistry, synthetic chemistry, biochemistry, and chemical biology to identify the substrates of peptidases in vivo. To accomplish this we utilize a liquid chromatography-mass spectrometry (LC-MS)-based peptidomics platform to measure changes in the peptidome as a function of peptidase activity. Previous analysis of mice lacking the enzyme dipeptidyl peptidase 4 (DPP4(-/-) mice), a biomedically relevant peptidase, using this approach identified a handful of novel endogenous DPP4 substrates. Here, we utilize these substrates and tissues from DPP4(-/-) mice to improve the coverage of the peptidomics platform by optimizing the key steps in the workflow, and in doing so, discover over 70 renal DPP4 substrates (up from 7 at the beginning of our optimization), a 10-fold improvement in our coverage. The sequences of these DPP4 peptide substrates support a broad role for DPP4 in proline-containing peptide catabolism and strengthen a biochemical model that interlinks aminopeptidase and DPP4 activities. Moreover, the improved peptidome coverage also led to the detection of greater numbers of known bioactive peptides (e.g., peptide hormones) during the analysis of gut samples, suggesting additional uses for this optimized workflow. Together these results strengthen our ability to identify endogenous peptide substrates through improved peptidome coverage and demonstrate a broader potential of this peptidomics platform.

Using X-ray Diffraction Techniques for Biomimetic Drug Development, Formulation, and Polymorphic Characterization.

Drug development is a decades-long, multibillion dollar investment that often limits itself. To decrease the time to drug approval, efforts are focused on drug targets and drug formulation for optimal biocompatibility and efficacy. X-ray structural characterization approaches have catalyzed the drug discovery and design process. Single crystal X-ray diffraction (SCXRD) reveals important structural details and molecular interactions for the manifestation of a disease or for therapeutic effect. Powder X-ray diffraction (PXRD) has provided a method to determine the different phases, purity, and stability of biological drug compounds that possess crystallinity. Recently, synchrotron sources have enabled wider access to the study of noncrystalline or amorphous solids. One valuable technique employed to determine atomic arrangements and local atom ordering of amorphous materials is the pair distribution function (PDF). PDF has been used in the study of amorphous solid dispersions (ASDs). ASDs are made up of an active pharmaceutical ingredient (API) within a drug dispersed at the molecular level in an amorphous polymeric carrier. This information is vital for appropriate formulation of a drug for stability, administration, and efficacy purposes. Natural or biomimetic products are often used as the API or the formulation agent. This review profiles the deep insights that X-ray structural techniques and associated analytical methods can offer in the development of a drug.