Solution Structure And Constrained Molecular Dynamics Study Of Vitamin B12 Conjugates Of The Anorectic Peptide PYY(3-36)

#50, published in ChemMedChem (11 (2016), 9, 1015-1020), DOI:cmdc.201600073.

The key to molecular dynamics simulations is recycling – specifically, going into a first project with enough organization to know how to use everything in the next study. While that first successful connectivity table, parameter assignment, and RESP charge generation for something as Frankenstein-esque as vitamin B12 is the north face of Everest, that next simulation is simply a matter of having atom codes in your PDB file standardized.

And, speaking of PDBs, article #50 has the added bonus of having its own entry in the Protein Databank as 2NA5 – quite a treat (to me, anyway).

And furthermore, this is the first of my publications to benefit from the Research Computing infrastructure on the Syracuse University campus – the throughput of calculations for future work is completely unprecedented in my history of resource access anywhere (the drop in storage prices is very real to some of us).


Authors: Henry K.E., Kerwood D.J., Allis D.G., Workinger J.L., Bonaccorso R.L., Holz G.G., Roth C.L., Zubieta J., and Doyle R.P.

Abstract: Vitamin B12–peptide conjugates have considerable therapeutic potential through improved pharmacokinetic and/or pharmacodynamic properties imparted on the peptide upon covalent attachment to vitamin B12 (B12). There remains a lack of structural studies investigating the effects of B12 conjugation on peptide secondary structure. Determining the solution structure of a B12–peptide conjugate or conjugates and measuring functions of the conjugate(s) at the target peptide receptor may offer considerable insight concerning the future design of fully optimized conjugates. This methodology is especially useful in tandem with constrained molecular dynamics (MD) studies, such that predictions may be made about conjugates not yet synthesized. Focusing on two B12 conjugates of the anorectic peptide PYY(3–36), one of which was previously demonstrated to have improved food intake reduction compared with PYY(3–36), we performed NMR structural analyses and used the information to conduct MD simulations. The study provides rare structural insight into vitamin B12 conjugates and validates the fact that B12 can be conjugated to a peptide without markedly affecting peptide secondary structure.

ChemMedChem Cover For April 2013 – Treating Type II Diabetes Through B12 Conjugation

The back cover picture shows two views at 150 degree rotation of vitamin B12 conjugated to the potent anti- hyperglycemia peptide glucagon-like peptide-1 (GLP-1). The conjugate displays similar receptor binding and agonism to unconjugated GLP-1, including insulin potentiation from human transplant pancreatic islet cells, which bodes well for oral delivery of GLP-1 through the B12 dietary pathway. For more details, see the Communication by Robert P. Doyle et al. on p. 582 ff.

From the free press department… The cover for the April, 2013 issue of ChemMEDChem (just the cover art this time, no theoretical content in the associated article. All the theory’s figured out!). I’m still awaiting the journal’s posting of the article content but wanted to get something up in March. For related content, see the discussion on the “MedChemComm September 2012 Front Cover Image For The ‘Examining The Effects Of Vitamin B12 Conjugation…’ Paper” post or any of the B12-related posts on this site ( This work is similar in scope to the B12-insulin bioconjugate work in the previous studies, but now includes a different peptide (glucagon-like peptide-1) with similar properties.

Examining The Effects Of Vitamin B12 Conjugation On The Biological Activity Of Insulin: A Molecular Dynamic And In Vivo Oral Uptake Investigation

Published in MedChemComm (direct link: And Happy Belated New Year. After the methodological work that went into the Molecular Biosystems paper, this was a remarkably simple molecular dynamics study of the changes to vitamin B12 binding in transcobalamin II (TCII) with the B12 conjugated to the first amino acid side chain in the B-Chain of insulin. The structure of the B12-insulin conjugate is shown below in a molecular dynamics snapshot, which reveals that the binding of B12 to its TCII transport protein is negligibly affected.

And apparently the experiments went well, too. Cover hopefully to follow.

Susan Clardy-James, Damian G. Allis, Timothy J. Fairchild and Robert P. Doyle

Abstract: The practical use of the vitamin B12 uptake pathway to orally deliver peptides and proteins is much debated. To understand the full potential of the pathway however, a deeper understanding of the impact B12 conjugation has on peptides and proteins is needed. We previously reported an orally active B12 based insulin conjugate attached at LysB29 with hypoglycaemic properties in STZ diabetic rats. We are exploring an alternative attachment for B12 on insulin in an attempt to determine the effect B12 has on the protein biological activity. We describe herein the synthesis, characterization, and purification of a new B12-insulin conjugate, which is attached between the B12 ribose hydroxyl group and insulin PheB1. The hypoglycemic properties resulting from oral administration (gavage) of such a conjugate in STZ diabetic rats was similar to that noted in a conjugate covalently linked at insulin LysB2911, demonstrating the availability of both position on insulin for B12 attachment. A possible rationale for this result is put forward from MD simulations. We also conclude that there is a dose dependent response that can be observed for B12-insulin conjugates, with doses of conjugate greater than 10-9 M necessary to observe even low levels of glucose drop.

New B12-Insulin-TCII-Insulin Receptor Cover Image For This Month’s ChemMedChem (March 2009)

As was the case for the first ChemMedChem December, 2007 cover issue (posted previously), the cover story in this month’s issue is a communication by myself and members and collaborators of the Robert Doyle Group here at Syracuse University.  In this case, the work for the cover image actually went into computational research published in the associated article (instead of just a pretty cover image to complement the associated article, which was the intent of the previous cover).

The image below shows the Transcobalamin II (TCII) protein (in teal ribbons, with a bound cyanocobalamin (B12) shown in red.  The PDB code for this complex is 2BB5) sitting within the surface-accessible fragment of the gigantic insulin receptor (PDB code 2DTG.  The cell membrane would be at the bottom of this image, with the remainder of the complete protein sitting both within the cell membrane and then into the cytoplasm).  Saving the lead-up to this structure generation for the associated published article, this image was created to show one of the most important steps in the Oral Insulin project being worked on in the Doyle Group, with the fact that we know it works making the validity of the image content all the more relevant.  In brief, this figure shows that the TCII/B12-Insulin complex can fit within the insulin receptor such that the insulin molecule can bind to its receptor position on the appropriately described insulin receptor (IR), thereby instigating the cascade of events that leads to cellular glucose uptake.

For a larger view, click on the image.

Continue reading “New B12-Insulin-TCII-Insulin Receptor Cover Image For This Month’s ChemMedChem (March 2009)”

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).