Thermodynamic consequences of stapling side-chains on a peptide ligand using a lactam-bridge: A theoretical study on anti-angiogenic peptides targeting VEGF.

Peptides are promising therapeutic agents for various biological targets due to their high efficacy and low toxicity, and the design of peptide ligands with high binding affinity to the target of interest is of utmost importance in peptide-based drug design. Introducing a conformational constraint to a flexible peptide ligand using a side-chain lactam-bridge is a convenient and efficient method to improve its binding affinity to the target. However, in general, such a small structural modification to a flexible ligand made with the intent of lowering the configurational entropic penalty for binding may have unintended consequences in different components of the binding enthalpy and entropy, including the configurational entropy component, which are still not clearly understood. Toward probing this, we examine different components of the binding enthalpy and entropy as well as the underlying structure and dynamics, for a side-chain lactam-bridged peptide inhibitor and its flexible analog forming complexes with vascular endothelial growth factor (VEGF), using all-atom molecular dynamics simulations. It is found that introducing a side-chain lactam-bridge constraint into the flexible peptide analog led to a gain in configurational entropy change but losses in solvation entropy, solute internal energy, and solvation energy changes upon binding, pinpointing the opportunities and challenges in drug design. The present study features an interplay between configurational and solvation entropy changes, as well as the one between binding enthalpy and entropy, in ligand-target binding upon imposing a conformational constraint into a flexible ligand.

An Ensemble Docking Approach for Analyzing and Designing Aptamer Heterodimers Targeting VEGF165.

Vascular endothelial growth factor 165 (VEGF165) is a prominent isoform of the VEGF-A protein that plays a crucial role in various angiogenesis-related diseases. It is homodimeric, and each of its monomers is composed of two domains connected by a flexible linker. DNA aptamers, which have emerged as potent therapeutic molecules for many proteins with high specificity and affinity, can also work for VEGF165. A DNA aptamer heterodimer composed of monomers of V7t1 and del5-1 connected by a flexible linker (V7t1:del5-1) exhibits a greater binding affinity with VEGF165 compared to either of the two monomers alone. Although the structure of the complex formed between the aptamer heterodimer and VEGF165 is unknown due to the highly flexible linkers, gaining structural information will still be valuable for future developments. Toward this end of accessing structural information, we adopt an ensemble docking approach here. We first obtain an ensemble of structures for both VEGF165 and the aptamer heterodimer by considering both small- and large-scale motions. We then proceed through an extraction process based on ensemble docking, molecular dynamics simulations, and binding free energy calculations to predict the structures of the VEGF165/V7t1:del5-1 complex. Through the same procedures, we reach a new aptamer heterodimer that bears a locked nucleic acid-modified counterpart of V7t1, namely RNV66:del5-1, which also binds well with VEGF165. We apply the same protocol to the monomeric units V7t1, RNV66, and del5-1 to target VEGF165. We observe that V7t1:del5-1 and RNV66:del5-1 show higher binding affinities with VEGF165 than any of the monomers, consistent with experiments that support the notion that aptamer heterodimers are more effective anti-VEGF165 aptamers than monomeric aptamers. Among the five different aptamers studied here, the newly designed RNV66:del5-1 shows the highest binding affinity with VEGF165. We expect that our ensemble docking approach can help in de novo designs of homo/heterodimeric anti-angiogenic drugs to target the homodimeric VEGF165.

Elucidating activation and deactivation dynamics of VEGFR-2 transmembrane domain with coarse-grained molecular dynamics simulations.

The vascular endothelial growth factor receptor 2 (VEGFR-2) is a member of receptor tyrosine kinases (RTKs) and is a dimeric membrane protein that functions as a primary regulator of angiogenesis. As is usual with RTKs, spatial alignment of its transmembrane domain (TMD) is essential toward VEGFR-2 activation. Experimentally, the helix rotations within TMD around their own helical axes are known to participate importantly toward the activation process in VEGFR-2, but the detailed dynamics of the interconversion between the active and inactive TMD forms have not been clearly elucidated at the molecular level. Here, we attempt to elucidate the process by using coarse grained (CG) molecular dynamics (MD) simulations. We observe that inactive dimeric TMD in separation is structurally stable over tens of microseconds, suggesting that TMD itself is passive and does not allow spontaneous signaling of VEGFR-2. By starting from the active conformation, we reveal the mechanism of TMD inactivation through analyzing the CG MD trajectories. We observe that interconversions between a left-handed overlay and a right-handed one are essential for the process of going from an active TMD structure to the inactive form. In addition, our simulations find that the helices can rotate properly when the overlaying structure of the helices interconverts and when the crossing angle of the two helices changes by larger than ~40 degrees. As the activation right after the ligand attachment on VEGFR-2 will take place in the reverse manner of this inactivation process, these structural aspects will also appear importantly for the activation process. The rather large change in helix configuration for activation also explains why VEGFR-2 rarely self-activate and how the activating ligand structurally drive the whole VEGFR-2. This mechanism of TMD activation / inactivation within VEGFR-2 may help in further understanding the overall activation processes of other RTKs.

Molecular mechanism of binding between a therapeutic RNA aptamer and its protein target VEGF: A molecular dynamics study.

Macugen is a therapeutic RNA aptamer against vascular endothelial growth factor (VEGF)-165, the VEGF isoform primarily responsible for angiogenesis. It has been reported that Macugen inhibits angiogenesis by specifically binding to the heparin binding domain (HBD) of VEGF165. The mechanism of the molecular recognition between HBD and Macugen is investigated here using all-atom molecular dynamics simulations. We find that Macugen recognizes HBD by an induced-fit mechanism with major conformational changes in Macugen and almost no changes in the structure of HBD, whereas HBD recognizes Macugen by a conformational selection mechanism.

Vectorial insertion of a ß-helical peptide into membrane: a theoretical study on polytheonamide B.

Spontaneous unidirectional, or vectorial, insertion of transmembrane peptides is a fundamental biophysical process for toxin and viral actions. Polytheonamide B (pTB) is a potent cytotoxic peptide with a ß6.3-helical structure. Previous experimental studies revealed that the pTB inserts into the membrane in a vectorial fashion and forms a channel with its single molecular length long enough to span the membrane. Also, molecular dynamics simulation studies demonstrated that the pTB is prefolded in aqueous solution. These are unique features of pTB because most of the peptide toxins form channels through oligomerization of transmembrane helices. Here, we performed all-atom molecular dynamics simulations to examine the dynamic mechanism of the vectorial insertion of pTB, providing underlying elementary processes of the membrane insertion of a prefolded single transmembrane peptide. We find that the insertion of pTB proceeds with only the local lateral compression of the membrane in three successive phases: "landing," "penetration," and "equilibration" phases. The free energy calculations using the replica-exchange umbrella sampling simulations present an energy cost of 4.3 kcal/mol at the membrane surface for the membrane insertion of pTB from bulk water. The trajectories of membrane insertion revealed that the insertion process can occur in two possible pathways, namely "trapped" and "untrapped" insertions; in some cases, pTB is trapped in the upper leaflet during the penetration phase. Our simulations demonstrated the importance of membrane anchoring by the hydrophobic N-terminal blocking group in the landing phase, leading to subsequent vectorial insertion.