Exploring the Implications of Vitamin B12 Conjugation to Insulin on Insulin Receptor Binding and Cellular Uptake

In press, in the journal ChemMedChem (and, because I think it’s hip, I note that the current “obligatory” image for the wikipedia article for ChemMedChem features the image I made for the review article on the topic addressed in this new study). As with many theory papers (there’s some experiment in there, too), this very brief article summarizes several months of cyanocobalamin (B12) parameterization and molecular dynamics (MD) simulations. The purpose of the theory was to address all of the major structural snapshots in the uptake process associated with the insulin-B12 bioconjugate being developed as part of the much heralded oral insulin project in Robert Doyle’s group here at Syracuse. These structures include:

1. The structure and dynamic properties of the insulin-B12 bioconjugate
2. The binding of B12 to Transcobalamin II (TCII) (for B12 parameterization)
3. The binding of the insulin-B12 bioconjugate to TCII (and the steric demands therein)
4. The interaction of the insulin-B12 bioconjugate, bound to TCII, with the insulin Receptor (IR)

The quantum chemical (for the B12 geometry and missing force constants) and molecular dynamics (GROMACS with the GROMOS96 (53a6)) simulation work is going to serve as the basis for several posts here (eventually) about parameterization, topology generation, and force field development.

As an example of some of the insights modeling provides, the figure above shows the insulin-B12 bioconjugate (the insulin is divided into A and B chains, the A chain in blue and the important division of the insulin B chain in the front half of the rainbow). Insulin is a rather large-scale example of many of the same molecular issues that arise in the analysis of solid-state molecular crystals by either terahertz or inelastic neutron scattering spectroscopy. The packing of molecules in their crystal lattices can lead to significant changes in molecular geometry, be these changes in the stabilization of higher-energy molecular conformations or even deformations in the covalent framework. In the case of insulin, it is found that the crystal geometry (also the geometry of stored insulin in the body) is quite different from the solution-phase form. It’s even worse! The B chain end (B20-B30) in the solid-state geometry covers (protects?) the business-end of the insulin binding region to the Insulin Receptor. One can imagine the difficulty in proposing the original binding model for insulin to its receptor from the original crystal data given that the actual binding region is blocked off in the solid-state form! The “Extended” form in the figure is representative of “multiple other” conformations of the B20-B30 region (which mimics the characterized T-state of insulin), those geometries for which the insulin binding region (blue and green) is completely exposed. This extended geometry is also the one that separates the bulk of the insulin structure from the covalently-linked B12 (at Lys29) and, it is argued from the MD simulations in the paper, enables the B12 to still tightly bind to TCII despite the presence of all this steric bulk.

Amanda K. Petrus1, Damian G. Allis1, Robert P. Smith2, Timothy J. Fairchild3 and Robert P. Doyle1

1. Department of Chemistry, Syracuse University, Syracuse, NY 13244, USA
2. Department of Construction Management and Wood Products Engineering, SUNY, College of Environmental Science and Forestry, Syracuse, NY 13210, USA
3. Department of Exercise Science, Syracuse University, Syracuse, NY 13244, USA

Extract: We recently reported a vitamin B12 (B12) based insulin conjugate that produced significantly decreased blood glucose levels in diabetic STZ-rat models. The results of this study posed a fundamental question, namely what implications does B12 conjugation have on insulin’s interaction with its receptor? To explore this question we used a combination of molecular dynamics (MD) simulations and immuno-electron microscopy (IEM).


Oral Insulin Delivery Cover Image (And Associated Syracuse Research Article) in ChemMedChem

You’ve heard about it, you’ve read about it, you’ve seen it on color TV, you’ve even seen it streamed. The cover story in this month’s issue of ChemMedChem is a communication by members and collaborators of the Robert Doyle Group here at Syracuse University. The report describes the B12/TCII-based uptake of insulin, a process that occurs via the ingestion of a B12-insulin conjugate. In case you missed that, the delivery is oral, not by needle. For those of us that pass out at anything needle-related at about the time that the alcohol wipe is opened, that’s a positive step forward for getting rid of any syringe-related medicine altogether.

full image

With the cover story comes the cover image shown above, a structure calculation on the insulin-B12/TCII complex. The bases for this structure can be found in the Protein Data Bank, including the TCII-B12 complex reported in PDB entry 2BB5 (the only hack in the structure calculation involved the replacement of the cobalt for iron to use already available bond parameters) and the insulin structure reported in PDB entry 1ZNI. The covalent attachment of the insulin to B12 can be found in the article. Structure manipulation was performed with a combination of NanoEngineer-1 and VMD, VMD being included in the mix in order to generate the ribbon renderings of the insulin and TCII protein backbones. As for the accuracy of the calculation, time and a synchrotron X-ray source will tell.

For much more information and numerous links to new stories related to the research in the article, I direct you to the group website of Robert Doyle and the various links to news stories available in his departmental publication list.

chemmedchem cover
From ChemMedChem. Click HERE to go to the article.

From the website:

Cover Picture: Vitamin B12 as a Carrier for the Oral Delivery of Insulin (ChemMedChem 12/2007). The cover picture shows an orally active, glucose-lowering vitamin B12-insulin conjugate bound to the B12 uptake protein transcobalamin II (TCII). The inset shows a close-up view of the TCII binding pocket. (Insulin is in red; vitamin B12 is in bright yellow.) For details, see the Communication by T. J. Fairchild, R. P. Doyle, et al. on p. 1717 ff.


Extension Of The Single Amino Acid Chelate Concept (SAAC) To Bifunctional Biotin Analogues For Complexation Of The M(CO)3+1 Core (M = Tc And Re): Syntheses, Characterization, Biotinidase Stability And Avidin Binding

In press, available from the journal Bioconjugate Chemistry. The modeling study for the avidinbiotin structure and the biotin derivatives were completed with the molecular dynamics program NAMD on a Dual G4/450 loaned to me from Apple for development work, for which I am grateful (I’ve performed molecular dynamics simulations with the Walrus). I did manage to smoke the motherboard during this experience, for which I apologize. Given the state of the machine after the autopsy, I’m hoping no one (especially Eric Zelman!) asks for it back, even when I’m 64.

I made mention of the reasons for some of this work in an interview I did for nanotech.biz, completely unrelate to the other content, in case anyone wants some background.

Shelly James, Kevin P. Maresca, Damian G. Allis, John F. Valliant, William Eckelman, John W. Babich, and Jon Zubieta

Abstract: Biotin and avidin form one of the most stable complexes known (KD = 10-15M-1) making this pairing attractive for a variety of biomedical applications including targeted radiotherapy. In this application one of the pair is attached to a targeting molecule while the other is subsequently used to deliver a radionuclide for imaging and/or therapeutic applications. Recently we reported a new single amino acid chelate (SAAC) capable of forming robust complexes with Tc(CO)3 or Re(CO)3 cores. We describe here the application of SAAC analogs for the development of a series of novel radiolabeled biotin derivatives capable of forming robust complexes with both Tc and Re. Compounds were prepared through varying modification of the free carboxylic acid group of biotin. Each 99mTc complex of SAAC-biotin was studied for their ability to bind avidin, susceptibility to biotinidase and specificity for avidin in an in vivo avidin-containing tumor model. The radiochemical stability of the 99mTc(CO)3 complexes was also investigated by challenging each 99mTc-complex with large molar excesses of cysteine and histidine at elevated temperature. All compounds were radiochemically stable for greater than 24 hours at elevated temperature in the presence of histidine and cysteine. Both [99mTc(CO)3(L6)]+1 [TcL6; L6 = biotinyl- amido- propyl- N,N- (dipicolyl)- amine] and [99mTc(CO)3(L12a)]+1 (TcL12; L12 = N,N-(dipicolyl)- biotin- amido- Boc- lysine; TcL12a; L12a = N,N- (dipicolyl)- biotin- amide- lysine) readily bound to avidin whereas [99mTc(CO)3(L9)]+1 [TcL9; L9 = N,N- (dipicolyl)- biotin- amine] demonstrated minimal specific binding. TcL6 and TcL9 were resistant to biotinidase cleavage while TcL12a, which contains a lysine linkage, was rapidly cleaved. The highest uptake in an in vivo avidin tumor model was exhibited by TcL6, followed by TcL9 and TcL12a, respectively. This is likely the result of both intact binding to avidin and resistance to circulating biotinidase. Ligand L6 is the first SAAC analogue of biotin to demonstrate potential as a radiolabeled targeting vector of biotin capable of forming robust radiochemical complexes with both 99mTc and rhenium radionuclides.Computational simulations were performed to assess biotin-derivative accommodation within the binding site of the avidin. These calculations demonstrate that deformation of the surface domain of the binding pocket can occur to accommodate the transition metal-biotin derivatives with negligible changes to the inner-β-barrel, the region most responsible for binding and retaining biotin and its derivatives.

P.S. This publication is also of some use for explaining the series of images on the current departmental brochure for the Syracuse U. Chemistry Department. Steric interactions affect the local geometries of protein binding pockets. And a good thing, too.

Click on the image for a larger version.