Exploratory studies on soluble small molecule CD4 mimics as HIV entry inhibitors.

Several small molecule CD4 mimics, which inhibit the interaction of gp120 with CD4, have been developed. Original CD4 mimics such as NBD-556, which has an aromatic ring, an oxalamide linker and a piperidine moiety, possess significant anti-HIV activity but with their hydrophobic aromatic ring-containing structures are poorly soluble in water. We have developed derivatives with a halopyridinyl group in place of the phenyl group, such as KKN-134, and found them to have excellent aqueous solubility. Other leads that were examined are YIR-821, a compound with a cyclohexane group in a spiro attachment to a piperidine ring and a guanidino group on the piperidine nitrogen atom, and its PEGylated derivative, TKB-002. YIR-821 and TKB-002 retain potent anti-HIV activity. Here, new CD4 mimics, in which the phenyl group was replaced by a halopyridinyl group with the halogen atoms in different positions, their derivatives without a cyclohexane group on the piperidine ring and their hybrid molecules with PEG units were designed and synthesized. Some of these compounds show significantly higher aqueous solubility with maintenance of certain levels of anti-HIV activity. The present data should be useful in the future design of CD4 mimic molecules.

Design, Synthesis, and Antiviral activity of 1,2,3,4-Tetrahydropyrimidine derivatives acting as novel entry inhibitors to target at "Phe43 cavity" of HIV-1 gp120.

The HIV-1 invasion is initiated with the interaction of viral glycoprotein gp120 and cellular receptor CD4. The binding mechanism reveals two major hotspots involved in gp120-CD4 interaction. The first one is a hydrophobic cavity (Phe43 cavity) on gp120 capped with phenyl ring of phe43CD4 and the second is the electrostatic interaction between positive charge of Arg59CD4 and negative charge of Asp368gp120. Targeting these hotspots, small molecules for entry inhibition and HIV-1 neutralization were designed and tested. In the process, pyrimidine derivatives were identified as potent molecules to intercept gp120-CD4 binding by targeting both the hotspots. Herein, the synthesis, characterization of 1,2,3,4-Tetrahydropyrimidine derivatives, and biological evaluation on 93IN101, a clade C virus are presented. The paper presents a novel set of entry inhibitors to target dual hotspots on gp120 to inhibit protein-protein interactions.

Systematic Evaluation of Fluorination as Modification for Peptide-Based Fusion Inhibitors against HIV-1 Infection.

With the emergence of novel viruses, the development of new antivirals is more urgent than ever. A key step in human immunodeficiency virus type 1 (HIV-1) infection is six-helix bundle formation within the envelope protein subunit gp41. Selective disruption of bundle formation by peptides has been shown to be effective; however, these drugs, exemplified by T20, are prone to rapid clearance from the patient. The incorporation of non-natural amino acids is known to improve these pharmacokinetic properties. Here, we evaluate a peptide inhibitor in which a critical Ile residue is replaced by fluorinated analogues. We characterized the influence of the fluorinated analogues on the biophysical properties of the peptide. Furthermore, we show that the fluorinated peptides can block HIV-1 infection of target cells at nanomolar levels. These findings demonstrate that fluorinated amino acids are appropriate tools for the development of novel peptide therapeutics.

Double Arylation of the Indole Side Chain of Tri- and Tetrapodal Tryptophan Derivatives Renders Highly Potent HIV-1 and EV-A71 Entry Inhibitors†.

We have recently described a new generation of potent human immunodeficiency virus (HIV) and EV-A71 entry inhibitors. The prototypes contain three or four tryptophan (Trp) residues bearing an isophthalic acid moiety at the C2 position of each side-chain indole ring. This work is now extended by both shifting the position of the isophthalic acid to C7 and synthesizing doubly arylated C2/C7 derivatives. The most potent derivative (50% effective concentration (EC50) HIV-1, 6 nM; EC50 EV-A71, 40 nM), 33 (AL-518), is a C2/C7 doubly arylated tetrapodal compound. Its superior anti-HIV potency with respect to the previous C2-arylated prototype is in consonance with its higher affinity for the viral gp120. 33 (AL-518) showed comparable antiviral activities against X4 and R5 HIV-1 strains and seems to interact with the tip and base of the gp120 V3 loop. Taken together, these findings support the interest in 33 (AL-518) as a useful new prototype for anti-HIV/EV71 drug development.

BMS Derivatives C7-Linked to ß-Cyclodextrin and Hyperbranched Polyglycerol Retain Activity against R5-HIV-1NLAD8 Isolates and Can Be Deemed Potential Microbicides.

Amides from indole-3-glyoxylic acid and 4-benzoyl-2-methylpiperazine, which are related to entry inhibitors developed by Bristol-Myers Squibb (BMS), have been synthesized with aliphatic chains located at the C7 position of the indole ring. These spacers contain an azido group suitable for the well-known Cu(I)-catalyzed (3+2)-cycloaddition or an activated triple bond for the nucleophilic addition of thiols under physiological conditions. Reaction with polyols (ß-cyclodextrin and hyperbranched polyglycerol) decorated with complementary click partners has afforded polyol-BMS-like conjugates that are not cytotoxic (TZM.bl cells) and retain the activity against R5-HIV-1NLAD8 isolates. Thus, potential vaginal microbicides based on entry inhibitors, which can be called of 4th generation, are reported here for the first time.

Hybrids of Small-Molecule CD4 Mimics with Polyethylene Glycol Units as HIV Entry Inhibitors.

CD4 mimics are small molecules that inhibit the interaction of gp120 with CD4. We have developed several CD4 mimics. Herein, hybrid molecules consisting of CD4 mimics with a long alkyl chain or a PEG unit attached through a self-cleavable linker were synthesized. In anti-HIV activity, modification with a PEG unit appeared to be more suitable than modification with a long alkyl chain. Thus, hybrid molecules of CD4 mimics, with PEG units attached through an uncleavable linker, were developed and showed high anti-HIV activity and low cytotoxicity. In investigation of pharmacokinetics in a rhesus macaque, a hybrid compound had a more effective PK profile than that of the parent compound, and intramuscular injection was a more useful administration route to maintain the high blood concentration of the CD4 mimic than intravenous injection. The presented hybrid molecules of CD4 mimics with a PEG unit would be practically useful when combined with a neutralizing antibody.

Design, synthesis, and antiviral activity of a series of CD4-mimetic small-molecule HIV-1 entry inhibitors.

We presented our continuing stride to optimize the second-generation NBD entry antagonist targeted to the Phe43 cavity of HIV-1 gp120. We have synthesized thirty-eight new and novel analogs of NBD-14136, earlier designed based on a CH2OH "positional switch" hypothesis, and derived a comprehensive SAR. The antiviral data confirmed that the linear alcohol towards the "N" (C4) of the thiazole ring yielded more active inhibitors than those towards the "S" (C5) of the thiazole ring. The best inhibitor, NBD-14273 (compound 13), showed both improved antiviral activity and selectivity index (SI) against HIV-1HXB2 compared to NBD-14136. We also tested NBD-14273 against a large panel of 50 HIV-1 Env-pseudotyped viruses representing clinical isolates of diverse subtypes. The overall mean data indicate that antiviral potency against these isolates improved by ~3-fold, and SI also improved ~3-fold compared to NBD-14136. This new and novel inhibitor is expected to pave the way for further optimization to a more potent and clinically relevant inhibitor against HIV-1.

Design, synthesis and biological evaluation of novel 5-((substituted quinolin-3-yl/1-naphthyl) methylene)-3-substituted imidazolidin-2,4-dione as HIV-1 fusion inhibitors.

A series of novel 5-(substituted quinolin-3-yl or 1-naphthyl)methylene)-3-substituted imidazolidin-2,4-dione 9-26 was designed and synthesized. The prepared compounds were identified using 1H NMR, 13C NMR as well as elemental analyses. The inhibitory activity of 9-26 on HIV-1IIIB replication in MT-2 cells was evaluated. Some derivatives showed good to excellent anti-HIV activities as compounds 13, 18, 19, 20, 22 and 23. They showed EC50 of 0.148, 0.460, 0.332, 0.50, 0.271 and 0.420 µM respectively being more potent than compound I (EC50 = 0.70 µM) and II ( EC50 = 2.40 µM) as standards. The inhibitory activity of 9-26 on infected primary HIV-1 domain, 92US657 (clade B, R5) was investigated. All the tested compounds consistently inhibited infection of this virus with EC50 from 0.520 to 11.857 µM. Results from SAR studies showed that substitution on ring A with 6/7/8-methyl group resulted in significant increase in the inhibitory activity against HIV-1IIIB infection (5- >300 times) compared to the unsubstituted analog 9. The cytotoxicity of these compounds on MT-2 cells was tested and their CC50 values ranged from 11 to 85 µM with selectivity indexes ranged from 0.53 to 166. The docking study revealed nice fitting of the new compounds into the hydrophobic pocket of HIV-1 gp41 and higher affinity than NB-64. Compound 13, the most active in preventing HIV-1IIIB infection, adopted a similar orientation to compound IV. Molecular docking analysis of the new compounds revealed hydrogen bonding interactions between the imidazolidine-2,4-dione ring and LYS574 which were missed in the weakly active derivatives.

Suitable fusion of N-terminal heptad repeats to achieve covalently stabilized potent N-peptide inhibitors of HIV-1 infection.

N-terminal heptad repeat (NHR)-derived peptide (N-peptide) fusion inhibitors, which are derived from human immunodeficiency virus (HIV) envelope glycoprotein 41 (gp41), are limited by aggregation and unstable trimer conformation. However, they could function as potent inhibitors of viral infection by forming a coiled-coil structure covalently stabilized by interchain disulfide bonds. We previously synthesized N-peptides with potent anti-HIV-1 activity and high stability by coiled-coil fusion and covalent stabilization. Here, we attempted to study the effects of NHRs of chimeric N-peptides by fusing de novo coiled-coil isopeptide bridge-tethered T21 peptides of different NHR lengths. Peptides (T21N23)3 and (T21N36)3 was a more potent HIV-1 fusion inhibitor than (T21N17)3. The site of isopeptide bond formation was precisely controlled and had little influence on N-peptide properties. The N-peptide (T21N36)3, which had a similar conformation as the NHR trimer and interacted well with the C34 peptide, may be useful for screening other C-peptides and small-molecule fusion inhibitors, and for studying the interactions between the NHR trimer and C-terminal heptad repeats.

The Tryptophan-Rich Motif of HIV-1 gp41 Can Interact with the N-Terminal Deep Pocket Site: New Insights into the Structure and Function of gp41 and Its Inhibitors.

Refolding of the HIV-1 gp41 N- and C-terminal heptad repeats (NHR and CHR, respectively) into a six-helix bundle (6-HB) juxtaposes viral and cellular membranes for fusion. The CHR-derived peptide T20 is the only clinically approved viral fusion inhibitor and has potent anti-HIV activity; however, its mechanism of action is not fully understood. In this study, we surprisingly found that T20 disrupted the α-helical conformation of the NHR-derived peptide N54 through its C-terminal tryptophan-rich motif (TRM) and that synthetic short peptides containing the TRM sequence, TRM8 and TRM12, disrupted the N54 helix in a dose-dependent manner. Interestingly, TRM8 efficiently interfered with the secondary structures of three overlapping NHR peptides (N44, N38, and N28) and interacted with N28, which contains mainly the deep NHR pocket-forming sequence, with high affinity, suggesting that TRM targeted the NHR pocket site to mediate the disruption. Unlike TRM8, the short peptide corresponding to the pocket-binding domain (PBD) of the CHR helix had no such disruptive effect, and the CHR peptide C34 could form a stable 6-HB with the NHR helix; however, addition of the TRM to the C terminus of C34 resulted in a peptide (C46) that destroyed the NHR helix. Although the TRM peptides alone had no anti-HIV activity and could not block the formation of 6-HB conformation, substitution of the TRM for the PBD in C34 resulted in a mutant inhibitor (C34TRM) with high binding and inhibitory capacities. Combined, the present data inform a new mode of action of T20 and the structure-function relationship of gp41.IMPORTANCE The HIV-1 Env glycoprotein mediates membrane fusion and is conformationally labile. Despite extensive efforts, the structural property of the native fusion protein gp41 is largely unknown, and the mechanism of action of the gp41-derived fusion inhibitor T20 remains elusive. Here, we report that T20 and its C-terminal tryptophan-rich motif (TRM) can efficiently impair the conformation of the gp41 N-terminal heptad repeat (NHR) coiled coil by interacting with the deep NHR pocket site. The TRM sequence has been verified to possess the ability to replace the pocket-binding domain of C34, a fusion inhibitor peptide with high anti-HIV potency. Therefore, our studies have not only facilitated understanding of the mechanism of action of T20 and developed novel HIV-1 fusion inhibitors but also provided new insights into the structural property of the prefusion state of gp41.

Long-Acting HIV-1 Fusion Inhibitory Peptides and their Mechanisms of Action.

The clinical application of HIV fusion inhibitor, enfuvirtide (T20), was limited mainly because of its short half-life. Here we designed and synthesized two PEGylated C34 peptides, PEG2kC34 and PEG5kC34, with the PEG chain length of 2 and 5 kDa, respectively, and evaluated their anti-HIV-1 activity and mechanisms of action. We found that these two PEGylated peptides could bind to the HIV-1 peptide N36 to form high affinity complexes with high α-helicity. The peptides PEG2kC34 and PEG5kC34 effectively inhibited HIV-1 Env-mediated cell-cell fusion with an effective concentration for 50% inhibition (EC50) of about 36 nM. They also inhibited infection of the laboratory-adapted HIV-1 strain NL4-3 with EC50 of about 4-5 nM, and against 47 HIV-1 clinical isolates circulating in China with mean EC50 of PEG2kC34 and PEG5kC34 of about 26 nM and 32 nM, respectively. The plasma half-life (t1/2) of PEG2kC34 and PEG5kC34 was 2.6 h and 5.1 h, respectively, and the t1/2 of PEGylated C34 was about 2.4-fold and 4.6-fold longer than C34 (~1.1 h), respectively. These findings suggest that PEGylated C34 with broad-spectrum anti-HIV-1 activity and prolonged half-life can be further developed as a peptide fusion inhibitor-based long-acting anti-HIV drug for clinical use to treat HIV-infected patients who have failed to respond to current anti-retrovirus drugs.

Revisiting the mechanism of enfuvirtide and designing an analog with improved fusion inhibitory activity by targeting triple sites in gp41.

OBJECTIVE: To revisit the mechanism of action of enfuvirtide (T20) and based on the newly defined mechanism, design an analogous peptide of T20 with improved antiviral activity. DESIGN: We compared the inhibitory activity of T20 with that of T1144 on six-helix bundle (6HB) formation at different time after coculture of HIV type 1 (HIV-1) envelope (Env)-expressing Chinese hamster ovary (CHO-Env) cells and CD4-expressing MT-2 cells at 31.5 °C and with that of T20-SF, an analogous peptide of T20 with an additional tryptophan-rich motif, on hemolysis mediated by FP-P, which contains fusion peptide and fusion peptide (FP) proximal region (FPPR), and HIV-1 infection. METHODS: Inhibitory activity of peptides on 6HB formation was tested in a temperature-controlled cell-cell fusion assay by flow cytometry using 6HB-specific mAb 2G8; on HIV-1 infection and fusion was assessed by p24 and cell-cell fusion assays. Interaction between different peptides or peptide and antibody was evaluated by ELISA. RESULTS: T20 could inhibit 6HB formation at early, but not late, stage of HIV-1 fusion, whereas T1144 was effective at both stages. T20-SF is much more effective than T20 in binding to FP-P and inhibiting infection of HIV-1, including T20-resistant strains, and FP-P-mediated hemolysis. CONCLUSION: Results suggest that T20 has a double-target mechanism, by which its N-terminal and C-terminal portions bind to N-terminal heptad repeat and FPPR, respectively. T20-SF designed based on this new mechanism exhibits significantly improved anti-HIV-1 activity because it targets the triple sites in gp41, including N-terminal heptad repeat, FPPR, and fusion peptide. Thus, this study provides clues for designing novel HIV fusion inhibitors with improved antiviral activity.

Dual activity of amphiphilic Zn(II) nitroporphyrin derivatives as HIV-1 entry inhibitors and in cancer photodynamic therapy.

Two Zn(II) nitro porphyrin derivatives bearing combinations of meso-4-nitrophenyl and meso-4-methylpyridinium moieties and their free-base precursors were synthesized through one-pot microwave process, purified and characterized. The biological activity of these nitroporphyrins was assessed under both photodynamic and non-photodynamic conditions to correlate their structure-activity relationship (SAR). Unlike, the free-base precursors, Zn(II) complexes of these nitroporphyrins displayed nearly complete inhibition in the entry of lentiviruses such as HIV-1 and SIVmac under non-photodynamic conditions. In addition, the Zn(II) complexes also exhibited a higher in vitro photodynamic activity towards human lung cancer cell-line A549 than their free-base precursors. Our results strongly suggest that incorporation of Zn(II) has improved the antiviral and anticancer properties of the nitroporphyrins. To the best of our knowledge, this is the first report demonstrating the dual activity of nitroporphyrin-zinc complexes as antiviral and anti-cancer, which will aid in their development as therapeutics in clinics.

Dimeric C34 Derivatives Linked through Disulfide Bridges as New HIV-1 Fusion Inhibitors.

C34, a 34-mer fragment peptide, is contained in the HIV-1 envelope protein gp41. A dimeric derivative of C34 linked through a disulfide bridge at its C terminus was synthesized and found to display potent anti-HIV activity, comparable with that of a previously reported PEGylated dimer of C34REG. The reduction in the size of the linker moiety for dimerization was thus successful, and this result might shed some light on the mechanism of the suppression of six-helix bundle formation by these C34 dimeric derivatives. Addition of a Gly-Cys(CH2 CONH2 )-Gly-Gly motif at the N-terminal position of a C34 monomeric derivative significantly increased the anti-HIV-1 activity. This moiety functions as a new pharmacophore, and this might provide a useful insight into the design of potent HIV-1 fusion inhibitors.

Investigation of the molecular characteristics of bisindole inhibitors as HIV-1 glycoprotein-41 fusion inhibitors.

In previous work, we described 6-6'-bisindole compounds targeting a hydrophobic pocket on the N-heptad repeat region of viral glycoprotein-41 as effective inhibitors of HIV-1 fusion. Two promising compounds with sub-micromolar IC50's contained a benzoic acid group and a benzoic acid ester attached at the two indole nitrogens. Here we have conducted a thorough structure-activity relationship (SAR) study evaluating the contribution of each of the ring systems and various substituents to compound potency. Hydrophobicity, polarity and charge were varied to produce 35 new compounds that were evaluated in binding, cell-cell fusion and viral infectivity assays. We found that (a) activity based solely on increasing hydrophobic content plateaued at ∼ 200 nM; (b) the bisindole scaffold surpassed other heterocyclic ring systems in efficacy; (c) a polar interaction possibly involving Gln575 in the pocket could supplant less specific hydrophobic interactions; and (d) the benzoic acid ester moiety did not appear to form specific contacts with the pocket. The importance of this hydrophobic group to compound potency suggests a mechanism whereby it might interact with a tertiary component during fusion, such as membrane. A promising small molecule 10b with sub-µM activity was discovered with molecular weight <500 da and reduced logP compared to earlier compounds. The work provides insight into requirements for small molecule inhibition of HIV-1 fusion.

Flexibility of small molecular CD4 mimics as HIV entry inhibitors.

CD4 mimics such as YIR-821 and its derivatives are small molecules which inhibit the interaction between the Phe43 cavity of HIV-1 gp120 with host CD4, an interaction that is involved in the entry of HIV to cells. Known CD4 mimics generally possess three structural features, an aromatic ring, an oxalamide linker and a piperidine moiety. We have shown previously that introduction of a cyclohexyl group and a guanidine group into the piperidine moiety and a fluorine atom at the meta-position of the aromatic ring leads to a significant increase in the anti-HIV activity. In the current study, the effects of conformational flexibility were investigated by introduction of an indole-type group in the junction between the oxalamide linker and the aromatic moiety or by replacement of the oxalamide linker with a glycine linker. This led to the development of compounds with high anti-HIV activity, showing the importance of the junction region for the expression of high anti-HIV activity. The present data are expected to be useful in the future design of novel CD4 mimic molecules.

Synthesis, biophysical characterization, and anti-HIV-1 fusion activity of DNA helix-based inhibitors with a p-benzyloxyphenyl substituent at the 5'-nucleobase site.

DNA helix-based HIV-1 fusion inhibitors have been discovered as potent drug candidates. Introduction of hydrophobic groups to a nucleobase provides an opportunity to design inhibitors with novel structures and mechanisms of action. In this work, two novel nucleoside analogues (1 and 2) were synthesized and incorporated into four DNA duplex- and quadruplex-based inhibitors. All the molecules showed anti-HIV-1 fusion activity. The effect of the p-benzyloxyphenyl group and the attached linker on the helix formation and thermal stability were fully compared and discussed. Surface plasmon resonance analysis further indicated that inhibitors with the same DNA helix may still have variable reaction targets, mainly attributed to the different hydrophobic modifications.

Pharmacokinetic and Chemical Synthesis Optimization of a Potent d-Peptide HIV Entry Inhibitor Suitable for Extended-Release Delivery.

Peptides often suffer from short in vivo half-lives due to proteolysis and renal clearance that limit their therapeutic potential in many indications, necessitating pharmacokinetic (PK) enhancement. d-Peptides, composed of mirror-image d-amino acids, overcome proteolytic degradation but are still vulnerable to renal filtration due to their small size. If renal filtration could be slowed, d-peptides would be promising therapeutic agents for infrequent dosing, such as in extended-release depots. Here, we tether a diverse set of PK-enhancing cargoes to our potent, protease-resistant d-peptide HIV entry inhibitor, PIE12-trimer. This inhibitor panel provides an opportunity to evaluate the PK impact of the cargoes independently of proteolysis. While all the PK-enhancing strategies (PEGylation, acylation, alkylation, and cholesterol conjugation) improved in vivo half-life, cholesterol conjugation of PIE12-trimer dramatically improves both antiviral potency and half-life in rats, making it our lead anti-HIV drug candidate. We designed its chemical synthesis for large-scale production (CPT31) and demonstrated that the PK profile in cynomolgous monkeys supports future development of monthly or less frequent depot dosing in humans. CPT31 could address an urgent need in both HIV prevention and treatment.

Synthesis and biological evaluation of water-soluble derivatives of chiral gossypol as HIV fusion inhibitors targeting gp41.

A series of novel or known water-soluble derivatives of chiral gossypol were synthesized and screened in vitro for their anti-HIV-1 activity. (-)-gossypol derivative was more active against HIV-1 than the corresponding (+)-gossypol derivative, respectively. Among these derivatives, d-glucosamine derivative of (-)-gossypol, oligopeptide derivative of (-)-gossypol and taurine derivative of (-)-gossypol, such as compounds 1a, 3a and 14a, showed significant inhibitory activities against HIV-1 replication, HIV-1 mediated cell-cell fusion and HIV gp41 6-helix bundle formation as some amino acid derivatives of (-)-gossypol.

Enhanced potency of bivalent small molecule gp41 inhibitors.

Low molecular weight peptidomimetic inhibitors with hydrophobic pocket binding properties and moderate fusion inhibitory activity against HIV-1 gp41-mediated cell fusion were elaborated by increasing the available surface area for interacting with the heptad repeat-1 (HR1) coiled coil on gp41. Two types of modifications were tested: 1) increasing the overall hydrophobicity of the molecules with an extension that could interact in the HR1 groove, and 2) forming symmetrical dimers with two peptidomimetic motifs that could potentially interact simultaneously in two hydrophobic pockets on the HR1 trimer. The latter approach was more successful, yielding 40-60times improved potency against HIV fusion over the monomers. Biophysical characterization, including equilibrium binding studies by fluorescence and kinetic analysis by Surface Plasmon Resonance, revealed that inhibitor potency was better correlated to off-rates than to binding affinity. Binding and kinetic data could be fit to a model of bidentate interaction of dimers with the HR1 trimer as an explanation for the slow off-rate, albeit with minimal cooperativity due to the highly flexible ligand structures. The strong cooperativity observed in fusion inhibitory activity of the dimers implied accentuated potency due to the transient nature of the targeted intermediate. Optimization of monomer, dimer or higher order structures has the potential to lead to highly potent non-peptide fusion inhibitors by targeting multiple hydrophobic pockets.