Extending Halogen-based Medicinal Chemistry to Proteins: IODO-INSULIN AS A CASE STUDY

Insulin, a protein critical for metabolic homeostasis, provides a classical model for protein design with application to human health. Recent efforts to improve its pharmaceutical formulation demonstrated that iodination of a conserved tyrosine (Tyr(B26)) enhances key properties of a rapid-acting clinical analog. Moreover, the broad utility of halogens in medicinal chemistry has motivated the use of hybrid quantum- and molecular-mechanical methods to study proteins. Here, we (i) undertook quantitative atomistic simulations of 3-[iodo-Tyr(B26)]insulin to predict its structural features, and (ii) tested these predictions by X-ray crystallography. Using an electrostatic model of the modified aromatic ring based on quantum chemistry, the calculations suggested that the analog, as a dimer and hexamer, exhibits subtle differences in aromatic-aromatic interactions at the dimer interface. Aromatic rings (Tyr(B16), Phe(B24), Phe(B25), 3-I-Tyr(B26), and their symmetry-related mates) at this interface adjust to enable packing of the hydrophobic iodine atoms within the core of each monomer. Strikingly, these features were observed in the crystal structure of a 3-[iodo-Tyr(B26)]insulin analog (determined as an R6 zinc hexamer). Given that residues B24-B30 detach from the core on receptor binding, the environment of 3-I-Tyr(B26) in a receptor complex must differ from that in the free hormone. Based on the recent structure of a "micro-receptor" complex, we predict that 3-I-Tyr(B26) engages the receptor via directional halogen bonding and halogen-directed hydrogen bonding as follows: favorable electrostatic interactions exploiting, respectively, the halogen's electron-deficient σ-hole and electronegative equatorial band. Inspired by quantum chemistry and molecular dynamics, such "halogen engineering" promises to extend principles of medicinal chemistry to proteins

A thing of beauty: Structure and function of insulin's "aromatic triplet"

The classical crystal structure of insulin was determined in 1969 by D.C. Hodgkin et al. following a 35-year program of research. This structure depicted a hexamer remarkable for its self-assembly as a zinc-coordinated trimer of dimer. Prominent at the dimer interface was an "aromatic triplet" of conserved residues at consecutive positions in the B chain: PheB24 , PheB25 and TyrB26 . The elegance of this interface inspired the Oxford team to poetry: "A thing of beauty is a joy forever" (John Keats as quoted by Blundell, T.L., et al. Advances in Protein Chemistry 26:279-286 [1972]). Here, we revisit this aromatic triplet in light of recent advances in the structural biology of insulin bound as a monomer to fragments of the insulin receptor. Such co-crystal structures have defined how these side chains pack at the primary hormone-binding surface of the receptor ectodomain. On receptor binding, the B-chain β-strand (residues B24-B28) containing the aromatic triplet detaches from the α-helical core of the hormone. Whereas TyrB26 lies at the periphery of the receptor interface and may functionally be replaced by a diverse set of substitutions, PheB24 and PheB25 engage invariant elements of receptor domains L1 and αCT. These critical contacts were anticipated by the discovery of diabetes-associated mutations at these positions by Donald Steiner et al. at the University of Chicago. Conservation of PheB24 , PheB25 and TyrB26 among vertebrate insulins reflects the striking confluence of structure-based evolutionary constraints: foldability, protective self-assembly and hormonal activity

Biosynthesis, structure, and folding of the insulin precursor protein

Insulin synthesis in pancreatic β-cells is initiated as preproinsulin. Prevailing glucose concentrations, which oscillate pre- and postprandially, exert major dynamic variation in preproinsulin biosynthesis. Accompanying upregulated translation of the insulin precursor includes elements of the endoplasmic reticulum (ER) translocation apparatus linked to successful orientation of the signal peptide, translocation and signal peptide cleavage of preproinsulin-all of which are necessary to initiate the pathway of proper proinsulin folding. Evolutionary pressures on the primary structure of proinsulin itself have preserved the efficiency of folding ("foldability"), and remarkably, these evolutionary pressures are distinct from those protecting the ultimate biological activity of insulin. Proinsulin foldability is manifest in the ER, in which the local environment is designed to assist in the overall load of proinsulin folding and to favour its disulphide bond formation (while limiting misfolding), all of which is closely tuned to ER stress response pathways that have complex (beneficial, as well as potentially damaging) effects on pancreatic β-cells. Proinsulin misfolding may occur as a consequence of exuberant proinsulin biosynthetic load in the ER, proinsulin coding sequence mutations, or genetic predispositions that lead to an altered ER folding environment. Proinsulin misfolding is a phenotype that is very much linked to deficient insulin production and diabetes, as is seen in a variety of contexts: rodent models bearing proinsulin-misfolding mutants, human patients with Mutant INS-gene-induced Diabetes of Youth (MIDY), animal models and human patients bearing mutations in critical ER resident proteins, and, quite possibly, in more common variety type 2 diabetes

Risk Factors and Biomarkers for Chronic Hepatitis B Associated Hepatocellular Carcinoma

Globally, hepatitis B virus (HBV) related hepatocellular carcinoma (HCC) is one of the major causes of cancer-related mortality. This is, in part, due to delayed diagnosis and limited therapeutic options with more advanced stages of the disease. Given the prognostic importance of early diagnosis, novel methods for early detection are in need. Unlike most other cancer types, tissue is not required to diagnose HCC and is frequently avoided given the inherent risks of liver biopsy, so less invasive methods of obtaining tumor material are currently under investigation. Material shed from tumors into the periphery are being investigated for their potential to both surveil and diagnose patients for HCC. These materials include circulating tumor cells, DNA, RNA, and exosomes, and are collectively termed a “liquid biopsy”. In this review article, we discuss the evolving literature regarding the different risk factors for HCC and the types of emerging novel biomarkers that show promise in the prevention and early diagnosis of HCC within the context of HBV infection

Risk Factors and Biomarkers for Chronic Hepatitis B Associated Hepatocellular Carcinoma

Globally, hepatitis B virus (HBV) related hepatocellular carcinoma (HCC) is one of the major causes of cancer-related mortality. This is, in part, due to delayed diagnosis and limited therapeutic options with more advanced stages of the disease. Given the prognostic importance of early diagnosis, novel methods for early detection are in need. Unlike most other cancer types, tissue is not required to diagnose HCC and is frequently avoided given the inherent risks of liver biopsy, so less invasive methods of obtaining tumor material are currently under investigation. Material shed from tumors into the periphery are being investigated for their potential to both surveil and diagnose patients for HCC. These materials include circulating tumor cells, DNA, RNA, and exosomes, and are collectively termed a “liquid biopsy”. In this review article, we discuss the evolving literature regarding the different risk factors for HCC and the types of emerging novel biomarkers that show promise in the prevention and early diagnosis of HCC within the context of HBV infection.</jats:p