Peptide Drug Design Using Alchemical Free Energy Calculation: An Application and Validation on Agonists of Ghrelin Receptor.

With recent large-scale applications and validations, the relative binding free energy (RBFE) calculated using alchemical free energy methods has been proven to be an accurate measure to probe the binding of small-molecule drug candidates. On the other hand, given the flexibility of peptides, it is of great interest to find out whether sufficient sampling could be achieved within the typical time scale of such calculation, and a similar level of accuracy could be reached for peptide drugs. However, the systematic evaluation of such calculations on protein-peptide systems has been less reported. Most reported studies of peptides were restricted to a limited number of data points or lacking experimental support. To demonstrate the applicability of the alchemical free energy method for protein-peptide systems in a typical real-world drug discovery project, we report an application of the thermodynamic integration (TI) method to the RBFE calculation of ghrelin receptor and its peptide agonists. Along with the calculation, the synthesis and in vitro EC50 activity of relamorelin and 17 new peptide derivatives were also reported. A cost-effective criterion to determine the data collection time was proposed for peptides in the TI simulation. The average of three TI repeats yielded a mean absolute error of 0.98 kcal/mol and Pearson's correlation coefficient (R) of 0.77 against the experimental free energy derived from the in vitro EC50 activity, showing good repeatability of the proposed method and a slightly better agreement than the results obtained from the arbitrary time frames up to 20 ns. Although it is limited by having one target and a deduced binding pose, we hope that this study can add some insights into alchemical free energy calculation of protein-peptide systems, providing theoretical assistance to the development of peptide drugs.

Discovery of a Double-Stapled Short Peptide as a Long-Acting HIV-1 Inactivator with Potential for Oral Bioavailability.

Different from most antiretroviral drugs that act as passive defenders to inhibit HIV-1 replication inside the host cell, virus inactivators can attack and inactivate HIV-1 virions without relying on their replication cycle. Herein, we describe the discovery of a hydrocarbon double-stapled helix peptide, termed D26. D26 is based on the HIV-1 gp41 protein lentiviral lytic peptide-3 motif (LLP3) sequence, which can efficiently inhibit HIV-1 infection and inactivate cell-free HIV-1 virions. It was noted that D26 was highly resistant to proteolytic degradation and exhibited a remarkably extended in vivo elimination half-life. Additionally, relative to its linear, nonstapled version, D26 exhibited much higher exposure in sanctuary sites for HIV-1. Amazingly, this lead compound also demonstrated detectable oral absorption. Thus, it can be concluded that D26 is a promising candidate for further development as a long-acting, orally applicable HIV-1 inactivator for the treatment of HIV-1 infection.

Study on In Vitro Metabolism and In Vivo Pharmacokinetics of Beauvericin.

Beauvericin (BEA) is a well-known mycotoxin produced by many fungi, including Beaveria bassiana. The purpose of this study was to evaluate the in vitro distribution and metabolism characteristics as well as the in vivo pharmacokinetic (PK) profile of BEA. The in vitro metabolism studies of BEA were performed using rat, dog, mouse, monkey and human liver microsomes, cryopreserved hepatocytes and plasma under conditions of linear kinetics to estimate the respective elimination rates. Additionally, LC-UV-MSn (n = 1~2) was used to identify metabolites in human, rat, mouse, dog and monkey liver microsomes. Furthermore, cytochrome P450 (CYP) reaction phenotyping was carried out. Finally, the absolute bioavailability of BEA was evaluated by intravenous and oral administration in rats. BEA was metabolically stable in the liver microsomes and hepatocytes of humans and rats; however, it was a strong inhibitor of midazolam 1'-hydroxylase (CYP3A4) and mephenytoin 4'-hydroxylase (CYP2C19) activities in human liver microsomes. The protein binding fraction values of BEA were >90% and the half-life (T1/2) values of BEA were approximately 5 h in the plasma of the five species. The absolute bioavailability was calculated to be 29.5%. Altogether, these data indicate that BEA has great potential for further development as a drug candidate. Metabolic studies of different species can provide important reference values for further safety evaluation.

Design and Biological Evaluation of m-Xylene Thioether-Stapled Short Helical Peptides Targeting the HIV-1 gp41 Hexameric Coiled-Coil Fusion Complex.

Short peptide-based inhibition of fusion remains an attractive goal in antihuman immunodeficiency virus (HIV) research based on its potential for the development of technically and economically desirable antiviral agents. Herein, we report the use of the dithiol bisalkylation reaction to generate a series of m-xylene thioether-stapled 22-residue α-helical peptides that have been identified as fusion inhibitors targeting HIV-1 glycoprotein 41 (gp41). The peptide sequence is based on the helix-zone binding domain of the gp41 C-terminal heptad repeat region. We found that one of these stapled peptides, named hCS6ERE, showed promising inhibitory potency against HIV-1 Env-mediated cell-cell fusion and viral replication at a level comparable to the clinically used 36-mer peptide T20. Furthermore, combining hCS6ERE with a fusion inhibitor having a different target site, such as HP23, produced synergistic anti-HIV-1 activity. Collectively, our study offers new insight into the design of anti-HIV peptides with short sequences.

Discovery of Hydrocarbon-Stapled Short α-Helical Peptides as Promising Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Fusion Inhibitors.

The hexameric α-helical coiled-coil formed between the C-terminal and N-terminal heptad repeat (CHR and NHR) regions of class I viral fusion proteins plays an important role in mediating the fusion of the viral and cellular membranes and provides a clear starting point for molecular mimicry that drives viral fusion inhibitor design. Unfortunately, such peptide mimicry of the short α-helical region in the CHR of Middle East respiratory syndrome coronavirus (MERS-CoV) spike protein has been thwarted by the loss of the peptide's native α-helical conformation when taken out of the parent protein structure. Here, we describe that appropriate all-hydrocarbon stapling of the short helical portion-based peptide to reinforce its bioactive secondary structure remarkably improves antiviral potency. The resultant stapled peptide P21S10 could effectively inhibit infection by MERS-CoV pseudovirus and its spike protein-mediated cell-cell fusion; additionally, P21S10 exhibits improved pharmacokinetic properties than HR2P-M2, suggesting strong potential for development as an anti-MERS-CoV therapeutic.

De Novo Design of α-Helical Lipopeptides Targeting Viral Fusion Proteins: A Promising Strategy for Relatively Broad-Spectrum Antiviral Drug Discovery.

Class I enveloped viruses share similarities in their apparent use of a hexameric coiled-coil assembly to drive the merging of virus and host cell membranes. Inhibition of coiled coil-mediated interactions using bioactive peptides that replicate an α-helical chain from the viral fusion machinery has significant antiviral potential. Here, we present the construction of a series of lipopeptides composed of a de novo heptad repeat sequence-based α-helical peptide plus a hydrocarbon tail. Promisingly, the constructs adopted stable α-helical conformations and exhibited relatively broad-spectrum antiviral activities against Middle East respiratory syndrome coronavirus (MERS-CoV) and influenza A viruses (IAVs). Together, these findings reveal a new strategy for relatively broad-spectrum antiviral drug discovery by relying on the tunability of the α-helical coiled-coil domains present in all class I fusion proteins and the amphiphilic nature of the individual helices from this multihelix motif.