Identification of Highly Potent Human Immunodeficiency Virus Type-1 Protease Inhibitors against Lopinavir and Darunavir Resistant Viruses from Allophenylnorstatine-Based Peptidomimetics with P2 Tetrahydrofuranylglycine.

The emergence of drug-resistant HIV from a widespread antiviral chemotherapy targeting HIV protease in the past decades is unavoidable and provides a challenge to develop alternative inhibitors. We synthesized a series of allophenylnorstatine-based peptidomimetics with various P3, P2, and P2́ moieties. The derivatives with P2 tetrahydrofuranylglycine (Thfg) were found to be potent against wild type HIV-1 protease and the virus, leading to a highly potent compound 21f (KNI-1657) against lopinavir/ritonavir- or darunavir-resistant strains. Co-crystal structures of 21f and the wild-type protease revealed numerous key hydrogen bonding interactions with Thfg. These results suggest that the strategy to design allophenylnorstatine-based peptidomimetics combined with Thfg residue would be promising for generating candidates to overcome multidrug resistance.

Cell-specific targeting by heterobivalent ligands.

Current cancer therapies exploit either differential metabolism or targeting to specific individual gene products that are overexpressed in aberrant cells. The work described herein proposes an alternative approach--to specifically target combinations of cell-surface receptors using heteromultivalent ligands ("receptor combination approach"). As a proof-of-concept that functionally unrelated receptors can be noncovalently cross-linked with high avidity and specificity, a series of heterobivalent ligands (htBVLs) were constructed from analogues of the melanocortin peptide ligand ([Nle(4), dPhe(7)]-α-MSH) and the cholecystokinin peptide ligand (CCK-8). Binding of these ligands to cells expressing the human Melanocortin-4 receptor and the Cholecystokinin-2 receptor was analyzed. The MSH(7) and CCK(6) were tethered with linkers of varying rigidity and length, constructed from natural and/or synthetic building blocks. Modeling data suggest that a linker length of 20-50 Å is needed to simultaneously bind these two different G-protein coupled receptors (GPCRs). These ligands exhibited up to 24-fold enhancement in binding affinity to cells that expressed both (bivalent binding), compared to cells with only one (monovalent binding) of the cognate receptors. The htBVLs had up to 50-fold higher affinity than that of a monomeric CCK ligand, i.e., Ac-CCK(6)-NH(2). Cell-surface targeting of these two cell types with labeled heteromultivalent ligand demonstrated high avidity and specificity, thereby validating the receptor combination approach. This ability to noncovalently cross-link heterologous receptors and target individual cells using a receptor combination approach opens up new possibilities for specific cell targeting in vivo for therapy or imaging.

Solid-Phase Synthesis of Heterobivalent Ligands Targeted to Melanocortin and Cholecystokinin Receptors.

Heteromultivalency provides a route to increase binding avidity and to high specificity when compared to monovalent ligands. The enhanced specificity can potentially serve as a unique platform to develop diagnostics and therapeutics. To develop new imaging agents based upon multivalency, we employed heterobivalent constructs of optimized ligands. In this report, we describe synthetic methods we have developed for the preparation of heterobivalent constructs consisting of ligands targeted simultaneously to the melanocortin receptor, hMC4R, and the cholecystokinin receptors, CCK-2R. Modeling data suggest that a linker distance span of 20-50 Å is needed to crosslink these two G-protein coupled receptors (GPCRs). The two ligands were tethered with linkers of varying rigidity and length, and flexible polyethylene glycol based PEGO chain or semi-rigid [poly(Pro-Gly)] linkers were employed for this purpose. The described synthetic strategy provides a modular way to assemble ligands and linkers on solid-phase supports. Examples of heterobivalent ligands are provided to illustrate the increased binding avidity to cells that express the complementary receptors.

Synthesis and evaluation of bivalent NDP-alpha-MSH(7) peptide ligands for binding to the human melanocortin receptor 4 (hMC4R).

We demonstrate the potential utility of multivalent ligands as targeting agents for cancer imaging or therapy by determining the binding of homobivalent ligands to their corresponding receptors. This manuscript details the synthesis and evaluation of a series of bivalent ligands containing two copies of the truncated heptapeptide version of [Nle4-D-Phe7]-alpha-melanocyte stimulating hormone (NDP-alpha-MSH), referred to as MSH(7). These were connected with various semirigid linkers containing Pro-Gly repeats, with or without flexible poly(ethylene glycol) (PEGO) moieties at their termini. Modeling data suggest a distance of 20-50 A between the ligand binding sites of two adjacent G-protein coupled receptors, GPCRs. These bivalent ligands were observed to bind with higher affinity compared to their monovalent counterparts. Data suggest these ligands may be capable of cross-linking adjacent receptors. An optimal linker length of 25 +/- 10 A, inferred from these ligands, correlated well with the inter-receptor distance estimated through modeling. Although there was no difference in maximal binding affinities between the ligands constructed with the Pro-Gly repeats versus those constructed with the PEGO inserts, the PEGO-containing ligands bound with high affinities over a greater range of linker lengths.

Squalene-derived flexible linkers for bioactive peptides.

A regiochemical and stereochemical mixture of flexible linkers bearing terminal azide functionality was synthesized in two steps from squalene and was used to connect two high affinity NDP-alpha-MSH ligands or two low affinity MSH(4) ligands. The ligands were N-terminally acylated using N-hydroxysuccinimidoyl 5-hexynoate and were subsequently attached to the linker via copper-catalyzed 'click' 3+2 cyclization of the azide and alkyne moieties. In vitro biological evaluations showed that the binding affinity to the human melanocortin 4 receptor was not diminished for most linker-ligand combinations relative to the corresponding parental ligand. Statistical and cooperative binding effects were observed for dimeric constructs containing the low affinity ligand MSH(4), but not for dimeric NDP-alpha-MSH constructs, presumably due to slow off rates for this high affinity ligand.

Design, synthesis, and validation of a branched flexible linker for bioactive peptides.

A branched flexible linker that incorporates a fluorescent dansyl moiety was synthesized and used to connect two high affinity NDP-alpha-MSH ligands or two low affinity MSH(4) ligands. The linker was incorporated into the conjugate by solid-phase synthesis. In vitro biological evaluations showed that potency of binding to the human melanocortin 4 receptor was not diminished for linker-ligand combinations relative to the corresponding ligand alone.