Shigeyoshi Harada

Enhanced exposure of human immunodeficiency virus type 1 primary isolate neutralization epitopes through binding of CD4 mimetic compounds

ABSTRACT N-(4-Chlorophenyl)-N′-(2,2,6,6-tetramethyl-piperidin-4-yl)-oxalamide (NBD-556) is a low-molecular-weight compound that reportedly blocks the interaction between human immunodeficiency virus type 1 (HIV-1) gp120 and its receptor CD4. We investigated whether the enhancement of binding of anti-gp120 monoclonal antibodies (MAbs) toward envelope (Env) protein with NBD-556 are similar to those of soluble CD4 (sCD4) by comparing the binding profiles of the individual MAbs to Env-expressing cell surfaces. In flow cytometric analyses, the binding profiles of anti-CD4-induced epitope (CD4i) MAbs toward NBD-556-pretreated Env-expressing cell surfaces were similar to the binding profiles toward sCD4-pretreated cell surfaces. To investigate the binding position of NBD-556 on gp120, we induced HIV-1 variants that were resistant to NBD-556 and sCD4 in vitro. At passage 21 in the presence of 50 μM NBD-556, two amino acid substitutions (S375N in C3 and A433T in C4) were identified. On the other hand, in the selection with sCD4, seven mutations (E211G, P212L, V255E, N280K, S375N, G380R, and G431E) appeared during the passages. The profiles of the mutations after the selections with NBD-556 and sCD4 were very similar in their three-dimensional positions. Moreover, combinations of NBD-556 with anti-gp120 MAbs showed highly synergistic interactions against HIV-1. We further found that after enhancing the neutralizing activity by adding NBD-556, the contemporaneous virus became highly sensitive to antibodies in the patients plasma. These findings suggest that small compounds such as NBDs may enhance the neutralizing activities of CD4i and anti-V3 antibodies in vivo.

CD4 mimics targeting the mechanism of HIV entry.

A structure-activity relationship study was conducted of several CD4 mimicking small molecules which block the interaction between HIV-1 gp120 and CD4. These CD4 mimics induce a conformational change in gp120, exposing its co-receptor-binding site. This induces a highly synergistic interaction in the use in combination with a co-receptor CXCR4 antagonist and reveals a pronounced effect on the dynamic supramolecular mechanism of HIV-1 entry.

CD4 mimics targeting the HIV entry mechanism and their hybrid molecules with a CXCR4 antagonist

Small molecules behaving as CD4 mimics were previously reported as HIV-1 entry inhibitors that block the gp120-CD4 interaction and induce a conformational change in gp120, exposing its co-receptor-binding site. A structure-activity relationship (SAR) study of a series of CD4 mimic analogs was conducted to investigate the contribution from the piperidine moiety of CD4 mimic 1 to anti-HIV activity, cytotoxicity, and CD4 mimicry effects on conformational changes of gp120. In addition, several hybrid molecules based on conjugation of a CD4 mimic analog with a selective CXCR4 antagonist were also synthesized and their utility evaluated.

Pathways for the emergence of multi-dideoxynucleoside-resistant HIV-1 variants.

Objective: To investigate the mechanism by which the Q151M mutation in reverse transcriptase (RT) that confers multi-dideoxynucleoside resistance on HIV-1 and that requires a two base change (CAG←ATG) develops, and to understand the reason for the relatively lengthy period of time required for its emergence under therapy with multiple nucleoside RT inhibitors (NRTI). Design and methods: Propagation assays and competitive HIV-1 replication assays were used to evaluate the fitness of various infectious clones, including two putative intermediates (HIV-1Q151K(AAG) and HIV-1Q151L(CTG)) for HIV-1Q151M(ATG), in terms of sensitivity to zidovudine and didanosine. Steady-state kinetic constants of recombinant RT were also determined. Results: HIV-1Q151L replicated relatively poorly while HIV-1Q151K failed to replicate. When HIV-1Q151L was propagated further, it took three pathways in continuing to replicate: (i) HIV-1Q151L changed to HIV-1Q151M in eight of 16 experiments; (ii) HIV-1Q151L reverted to wild-type HIV-1 (HIV-1WT) in four of 16 experiments; and (iii) HIV-1Q151L acquired an additional mutation M230I in four of 16 experiments improving HIV-1 fitness. The relative order of replicative fitness without drugs was: HIV-1Q151M > HIV-1WT > HIV-1Q151L/M230I > HIV-1M230I >> HIV-1Q151L >>> HIV-1Q151K, HIV-1Q151K/M230I. HIV-1Q151M was less susceptible to drugs, while HIV-1Q151L/M230I was as sensitive as HIV-1WT. Enzymatic assays corroborated that HIV-1Q151L is more replication-competent than HIV-1WT and HIV-1Q151K in the presence of drugs. Conclusion: HIV-1Q151M probably develops through a poorly replicating HIV-1Q151L; however, it is also possible that it occurs through two concurrent base changes. The present data should explain the mechanism by which HIV-1Q151M emerges after long-term chemotherapy with NRTI.

Small molecular CD4 mimics as HIV entry inhibitors

Derivatives of CD4 mimics were designed and synthesized to interact with the conserved residues of the Phe43 cavity in gp120 to investigate their anti-HIV activity, cytotoxicity, and CD4 mimicry effects on conformational changes of gp120. Significant potency gains were made by installation of bulky hydrophobic groups into the piperidine moiety, resulting in discovery of a potent compound with a higher selective index and CD4 mimicry. The current study identified a novel lead compound 11 with significant anti-HIV activity and lower cytotoxicity than those of known CD4 mimics.

CD4 mimics as HIV entry inhibitors: Lead optimization studies of the aromatic substituents

Several CD4 mimics have been reported as HIV-1 entry inhibitors that can intervene in the interaction between a viral envelope glycoprotein gp120 and a cell surface protein CD4. Our previous SAR studies led to a finding of a highly potent analogue 3 with bulky hydrophobic groups on a piperidine moiety. In the present study, the aromatic ring of 3 was modified systematically in an attempt to improve its antiviral activity and CD4 mimicry which induces the conformational changes in gp120 that can render the envelope more sensitive to neutralizing antibodies. Biological assays of the synthetic compounds revealed that the introduction of a fluorine group as a meta-substituent of the aromatic ring caused an increase of anti-HIV activity and an enhancement of a CD4 mimicry, and led to a novel compound 13a that showed twice as potent anti-HIV activity compared to 3 and a substantial increase in a CD4 mimicry even at lower concentrations.

Identification of Amino Acid Residues Critical for LD78β, a Variant of Human Macrophage Inflammatory Protein-1α, Binding to CCR5 and Inhibition of R5 Human Immunodeficiency Virus Type 1 Replication

In an attempt to determine which amino acid(s) of LD78β, a variant of human macrophage inflammatory protein-1α, plays a critical role in the interaction with CCR5, we generated six LD78β variants with an amino acid substituted to Ala at the NH2 terminus of LD78β. There was no significant difference in eliciting Ca2+ flux and chemotaxis among the variants with the exception of LD78βT9A showing a substantially reduced activity. The comparative order for human immunodeficiency virus type 1 (HIV-1) replication inhibition was: LD78βP8A > LD78βD6A > LD78βWT, LD78βL3A > LD78βT7A, LD78βP2A > LD78βT9A. In binding inhibition assays of LD78β variants using 2D7 monoclonal antibody and 125I-labeled macrophage inflammatory protein-1α, the comparative order was: LD78βP8A, LD78βD6A > LD78βWT> LD78βL3A > LD78βT7A > LD78βT9A, LD78βP2A. The order for CCR5 down-regulation induction was comparable to that for binding inhibition. The present data suggest that Pro-2, Asp-6, Pro-8, and Thr-9 are critical for LD78β binding to CCR5 and HIV-1 replication inhibition, and that LD78β binding to CCR5, regardless of affinity, is sufficient for the initial signal transduction of LD78β, whereas the greater anti-HIV-1 activity requires the greater magnitude of binding. The data also suggest that LD78β variants with appropriate amino acid substitution(s) such as LD78βD6Aand LD78βP8A may represent effective chemokine-based anti-HIV-1 therapeutics while preserving LD78β-CCR5 interactions.

Impact of maraviroc-resistant and low-CCR5-adapted mutations induced by in vitro passage on sensitivity to anti-envelope neutralizing antibodies.

The aim of this study was to generate maraviroc (MVC)-resistant viruses in vitro using a human immunodeficiency virus type 1 subtype B clinical isolate (HIV-1KP-5) to understand the mechanism(s) of resistance to MVC. To select HIV-1 variants resistant to MVC in vitro, we exposed high-chemokine (C-C motif) receptor 5 (CCR5)-expressing PM1/CCR5 cells to HIV-1KP-5 followed by serial passage in the presence of MVC. We also passaged HIV-1KP-5 in PM1 cells, which were low CCR5 expressing to determine low-CCR5-adapted substitutions and compared the Env sequences of the MVC-selected variants. Following 48 passages with MVC (10 µM), HIV-1KP-5 acquired a resistant phenotype [maximal per cent inhibition (MPI) 24%], whilst the low-CCR5-adapted variant had low sensitivity to MVC (IC50 ~200 nM), but not reduction of the MPI. The common substitutions observed in both the MVC-selected and low-CCR5-adapted variants were selected from the quasi-species, in V1, V3 and V5. After 14 passages, the MVC-selected variants harboured substitutions around the CCR5 N-terminal-binding site and V3 (V200I, T297I, K305R and M434I). The low-CCR5-adapted infectious clone became sensitive to anti-CD4bs and CD4i mAbs, but not to anti-V3 mAb and autologous plasma IgGs. Conversely, the MVC-selected clone became highly sensitive to the anti-envelope (Env) mAbs tested and the autologous plasma IgGs. These findings suggest that the four MVC-resistant mutations required for entry using MVC-bound CCR5 result in a conformational change of Env that is associated with a phenotype sensitive to anti-Env neutralizing antibodies.

A CD4 mimic as an HIV entry inhibitor: Pharmacokinetics

To date, several small molecules of CD4 mimics, which can suppress competitively the interaction between an HIV-1 envelope glycoprotein gp120 and a cellular surface protein CD4, have been reported as viral entry inhibitors. A lead compound 2 (YYA-021) with relatively high potency and low cytotoxicity has been identified previously by SAR studies. In the present study, the pharmacokinetics of the intravenous administration of compound 2 in rats and rhesus macaques is reported. The half-lives of compound 2 in blood in rats and rhesus macaques suggest that compound 2 shows wide tissue distribution and relatively high distribution volumes. A few hours after the injection, both plasma concentrations of compound 2 maintained micromolar levels, indicating it might have promise for intravenous administration when used combinatorially with anti-gp120 monoclonal antibodies.

Small-Molecule CD4 Mimics Containing Mono-cyclohexyl Moieties as HIV Entry Inhibitors.

CD4 mimics are small molecules that inhibit the protein–protein interaction between gp120 and CD4, which is a key interaction for the entry of human immunodeficiency virus (HIV) into host immune cells. In the present study, mono‐cyclohexyl‐type CD4 mimics were designed to form hydrophobic and electrostatic interactions with Val430 and Asp368 located in the entrance of the Phe43 cavity of gp120, the interaction site of CD4. YIR‐329, a novel 1‐azaspiro[5.5]undecane derivative with a cyclohexyl ring attached to the piperidine ring, exhibited only slightly weaker anti‐HIV activity than a previously described lead HAR‐171, and modeling results indicated the formation of advantageous interactions by the para‐chlorophenyl moiety of YIR‐329. To introduce an electrostatic interaction with Asp368, derivatives with a guanidino group on the piperidine nitrogen atom were synthesized. Mono‐cyclohexyl‐type CD4 mimics with a guanidino group, such as YIR‐819 (N1‐(4‐chlorophenyl)‐N2‐(1‐(2‐(N‐(amidino)glycinamide)ethyl)‐2‐cyclohexylpiperidin‐4‐yl)oxalamide) and YIR‐821 (1‐(2‐(5‐guanidinovaleramide)ethyl derivative of YIR‐819), were identified that exhibit approximately fivefold more potent anti‐HIV activity than YIR‐329. In combination with a neutralizing antibody, their anti‐HIV activities were augmenting. Modeling results suggest that these compounds interact effectively with Val430 and either Asp368 or Asp474 in the gp120 Phe43 cavity. YIR‐819 and YIR‐821 represent useful lead compounds for the further development of HIV‐1 entry inhibitors and could potentially be useful for co‐administration with neutralizing antibodies for the treatment of HIV infection and AIDS.