Resistance profiles of novel electrostatically constrained HIV-1 fusion inhibitors.

Human immunodeficiency virus (HIV) gp41 plays a key role in viral fusion; the N- and C-terminal heptad repeats (N-HR and C-HR) of gp41 form a stable 6-helical conformation for fusion. Therefore, HR-derived peptides, such as enfuvirtide (T-20), inhibit HIV-1 fusion by acting as decoys, and have been used for the treatment of HIV-1 infection. However, the efficacy of T-20 is attenuated by resistance mutations in gp41, including V38A and N43D. To suppress the resistant variants, we previously developed electrostatically constrained peptides, SC34 and SC34EK, and showed that both exhibited potent anti-HIV-1 activity against wild-type and T-20-resistant variants. In this study, to clarify the resistance mechanism to this next generation of fusion inhibitors, we selected variants with resistance to SC34 and SC34EK in vitro. The resistant variants had multiple mutations in gp41. All of these mutations individually caused less than 6-fold resistance to SC34 and SC34EK, indicating that there is a significant genetic barrier for high-level resistance. Cross-resistance to SC34 and SC34EK was reduced by a simple difference in the polarity of two intramolecular electrostatic pairs. Furthermore, the selected mutations enhanced the physicochemical interactions with N-HR variants and restored activities of the parental peptide, C34, even to resistant variants. These results demonstrate that our approach of designing gp41-binding inhibitors using electrostatic constraints and information derived from resistance studies produces inhibitors with enhanced activity, high genetic barrier, and distinct resistance profile from T-20 and other inhibitors. Hence, this is a promising approach for the design of future generation peptide fusion inhibitors.

Mutations conferring resistance to human immunodeficiency virus type 1 fusion inhibitors are restricted by gp41 and Rev-responsive element functions.

One of the human immunodeficiency virus (HIV) envelope proteins, gp41, plays a key role in HIV fusion. A gp41-derived peptide, T-20, efficiently inhibits HIV fusion and is currently approved for treatment of HIV-infected individuals. Although resistant variants have been reported, the mechanism of the resistance remains to be defined. To elucidate the mechanism in detail, we generated variants resistant to C34, a peptide derived from the gp41 carboxyl terminus heptad repeat (C-HR) in vitro. The resistant variants had a 5-amino-acid deletion in gp120 and a total of seven amino acid substitutions in gp41. Binding assays revealed that an I37K substitution in the N-terminal heptad repeat (N-HR) impaired the binding of C34, whereas an N126K substitution in the C-HR enhanced the binding to mutated N-HR, indicating that both mutations were directly involved in resistance. On the other hand, substitutions for A30 and D36 seemed to be secondary mutations, located complementary to each other in the Rev-responsive element (RRE), and were mutated simultaneously to maintain the secondary structure of the RRE that was impaired by the mutations at I37. Thus, HIV acquired resistance to C34 by mutations in N-HR, which directly interacted with C34. However, since this region also encoded the RRE, additional mutations were required to maintain viral replication. These results suggest that HIV fusion is one of the attractive targets for HIV chemotherapy.

RNase S complex bearing arginine-rich peptide and anti-HIV activity.

Basic peptide-mediated protein delivery into living cells is becoming recognized as a potent approach for the understanding of cellular mechanisms and drug delivery. We have prepared the conjugates of the S-peptide (1-15) derived from RNase S with membrane-permeable basic peptides, octaarginine and the human immunodeficient virus (HIV)-1 Rev (34-50). The RNase S complexes, formed among these S-peptide (1-15)-basic peptide conjugates and the S-protein and having a dissociation constant in the range of 10(-5) M, efficiently penetrated into the HeLa cells. These RNase S complexes exerted an anti-HIV replication activity. The time-of-drug-addition assay suggested that the site of action for these complexes would reside in the stages between the viral entry into the cells and reverse transcription. The present study exemplified the applicability of the arginine-rich peptides to the intracellular targeting of non-covalent protein complexes and supramolecular assemblies for the research in chemical and cellular biology.

Attempt to reduce cytotoxicity by synthesizing the L-enantiomer of 4'-C-ethynyl-2'-deoxypurine nucleosides as antiviral agents against HIV and HBV.

We investigated the potential of 4'-C-substituted nucleosides for the treatment of HIV-1 and HBV. Of the nucleosides we prepared, several 4'-C-ethynyl-2'-deoxypurine nucleosides showed the most potent anti-HIV activity. However, two candidates, 4'-C-ethynyl-2'-deoxyguanosine and 9-(2-deoxy-4-C-ethynyl-beta-D-ribo-pentofuranosyl)-2,6-diaminopurine, were very toxic during in vivo study. On the other hand, lamivudine (3TC) is known to show remarkable activity against HIV and HBV with lower cytotoxicity. Therefore, we attempted to synthesize the L-enantiomer of 4'-C-ethynyl-2'-deoxypurine nucleosides in 20-21 steps. These methods consisted of preparing 4-C-ethynyl-L-sugar, starting from D-arabinose and then condensing the L-sugar derivative with 2,6-diaminopurine. 4'-C-Ethynyl-2'-deoxyguanosine was also prepared by enzymatic deamination from the 2,6-diaminopurine derivative. The compounds' antiviral activity against HIV and HBV was then evaluated. Unfortunately, they demonstrated no activity and no cytotoxicity.

Design, efficient synthesis, and anti-HIV activity of 4'-C-cyano- and 4'-C-ethynyl-2'-deoxy purine nucleosides.

Some 4'-C-ethynyl-2'-deoxy purine nucleosides showed the most potent anti-HIV activity among the series of 4'-C-substituted 2'-deoxynucleosides whose 4'-C-substituents were methyl, ethyl, ethynyl and so on. Our hypothesis is that the smaller the substituent at the C-4' position they have, the more acceptable biological activity they show. Thus, 4'-C-cyano-2'-deoxy purine nucleosides, whose substituent is smaller than the ethynyl group, will have more potent antiviral activity. To prove our hypothesis, we planned to develop an efficient synthesis of 4'-C-cyano-2'-deoxy purine nucleosides (4'-CNdNs) and 4'-C-ethynyl-2'-deoxy purine nucleosides (4'-EdNs). Consequently, we succeeded in developing an efficient synthesis of six 2'-deoxy purine nucleosides bearing either a cyano or an ethynyl group at the C-4' position of the sugar moiety from 2'-deoxyadenosine and 2,6-diaminopurine 2'-deoxyriboside. Unfortunately, 4'-C-cyano derivatives showed lower activity against HIV-1, and two 4'-C-ethynyl derivatives suggested high toxicity in vivo.

Synthesis of 4'-C-ethynyl and 4'-C-cyano purine nucleosides from natural nucleosides and their anti-HIV activity.

Purine 2'-deoxynucleosides bearing an ethynyl or a cyano group at C-4' of the sugar moiety were synthesized from the corresponding 2'-deoxynucleosides. These compounds exhibited very potent anti-HIV activity, and remained active against drug resistant HIV strains.