Design, synthesis and biological evaluation of small molecule inhibitors of CD4-gp120 binding based on virtual screening

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Abstract

The low-molecular-weight compound JRC-II-191 inhibits infection of HIV-1 by blocking the binding of the HIV-1 envelope glycoprotein gp120 to the CD4 receptor and is therefore an important lead in the development of a potent viral entry inhibitor. Reported here is the use of two orthogonal screening methods, gold docking and ROCS shape-based similarity searching, to identify amine-building blocks that, when conjugated to the core scaffold, yield novel analogs that maintain similar affinity for gp120. Use of this computational approach to expand SAR produced analogs of equal inhibitory activity but with diverse capacity to enhance viral infection. The novel analogs provide additional lead scaffolds for the development of HIV-1 entry inhibitors that employ protein–ligand interactions in the vestibule of gp120 Phe 43 cavity.

Introduction

The acquired immunodeficiency syndrome (AIDS) is a consequence of the depletion of CD4+ lymphocytes caused by the human immunodeficiency virus (HIV-1) in an infected individual.1, 2 The infection is initiated by a series of attachment events, mediated by the HIV-1 viral spike which is comprised of two glycoproteins, gp120 and gp41.3 The attachment of HIV to the target T-cell lymphocyte occurs via binding of gp120 to the host CD4 receptor.4 CD4 binding causes a large conformational structuring of gp1205, 6 that forms and exposes the binding site for one of two chemokine receptors, either CCR5 or CXCR4.7 Chemokine receptor binding is the second obligatory event in viral entry,8, 9 and is followed by insertion of the gp41 fusion peptide into the host cell membrane,10 promoting viral and cell membrane fusion and viral entry.11

Several X-ray crystal structures of complexes of gp120 with CD4 and various human antibodies have been determined.12, 13 CD4 residues identified by mutagenesis and the crystal structure to be critical for binding to gp120 are Phe 43 and Arg 59.14 Residue Phe 43, located on the CD4 β-turn that contacts a large cavity that is formed upon gp120-CD4 binding, is flanked by the inner, outer and bridging sheet domains of gp120 (Fig. 1). Additionally, Arg59 forms an electrostatic interaction with Asp 368 on an adjacent α-helix.12, 15 Studies carried out by isothermal titration calorimetry show that CD4 binds to gp120 with large favorable enthalpy and unfavorable entropy changes (ΔH = −63 kcal/mol, −TΔS = 52 kcal/mol), observed for the binding of core gp120 proteins, as well as for the full-length gp120, to CD4, that can be attributed to the large conformational structuring of the previously unstructured regions in gp120.16, 17

The two compounds NBD-556 (41) and NBD-557,18 first discovered by Zhao et al. utilizing high-throughput screening, as well as analogs of these compounds prepared in our laboratory,19 have been shown to compete with CD4 binding. The chemotype of the NBD series consists of three essential regions: Region I, a para-substituted phenyl ring; Region II, an oxalamide linker; and Region III, a tetramethyl-piperidine (Fig. 2). Modification in Region I with various para-substitutions indicates that size and electron withdrawing character are determinants for binding affinity.19, 20 The Br and Cl para-substituted phenyl NBD compounds achieved the best binding affinity (c.f. Kd = 2.2 and 3.7 μM, respectively).24 Study of Region I meta-substitutions within the context of the p-Cl analog, indicates that a fluorine substituent is preferred over Cl, OH, CH3 or CF3 substitutions, whereas ortho substitution in Region I abolished the ability of the compounds to bind gp120 (Supplementary Tables 1–6). Further synthesis and testing of other diverse Region I analogs also suggested the low probability of enhancing binding affinity in the base of the Phe 43 cavity. Thus far, studies of para and meta-substituted NBD compounds with comparable binding affinities demonstrate a wide capacity to act as a CD4 antagonist (i.e., to inhibit HIV-infection of CD4+ cells) and as a CD4 agonist (i.e., to promote CCR5 binding and enhance viral infection in the absence of CD4).19 Assaying compounds on CD4-deficient Cf2Th-CCR5 cells to measure the enhancement of viral infection is a biological indicator of the extent to which a compound mimics CD4. Importantly, NBD mimicry of CD4-gp120 binding is further demonstrated by isothermal titration calorimetry (ITC), revealing a large unfavorable entropy change, compensated by a larger favorable change in enthalpy; thus, NBD analogs induce conformational changes in the HIV-1 envelope glycoproteins with a similar thermodynamic profile to those induced by CD4. Although CD4 mimics enhance viral infection in CD4-deficient cells by inducing gp120 conformational change, HIV-1 entry and infection is inhibited in CD4-expressing cells.19, 21 A recent study of this mechanism with NBD analog 1, (Table 1, Kd = 0.76 μM) demonstrates that HIV-1 infection is inhibited via induction of a short-lived activated state in addition to specific competition for CD4 binding.22

Changes to HIV gp120 residues (E370A, S375N) that flank the Phe 43 cavity confer resistance to NBD-556, supporting the model that NBD-556 binds within this cavity.23 Mutagenesis data and the SAR results of meta and para phenyl substitution of the NBD core, combined with molecular modeling,19, 20 support the hypothesis that the Region I aromatic ring binds in the base of the gp120 Phe 43 cavity. Studies of mutations in the vestibule of the Phe 43 cavity, however, had opposing effects: a change of aspartate to alanine at position 368 increased the binding affinity, enhancement of CD4 cell infection, and inhibition of CD4+ cell infection by 1; by contrast, decreases in affinity, viral enhancement in CD4 cells, and viral inhibition of CD4+ cells of 1 were observed when valine at position 430 was substituted with an alanine.19 This mutational data suggested that key protein–ligand interactions in the vestibule could be manipulated to optimize the inhibitory properties of the NBD compounds. A recent report exploring SAR of Region III also suggests the importance of the piperdine moiety to CD4 mimicry and anti-HIV activity.24

Inspired by the possibility of exploiting NBD interactions in the gp120 vestibule, in conjunction with the unmet need for CD4-gp120 inhibitors as therapeutic modalities to prevent HIV entry, we turned to NBD SAR results for further exploration. The goal was to identify new chemotypes of 1 that would maintain similar or enhanced gp120 binding affinity as measured by ITC, inhibit viral infection, and exhibit favorable pharmaceutical characteristics. We report here on Region I and II SAR and focus on protein–ligand interactions in Region III of 1, which presumably contacts the vestibule of the CD4-binding pocket (Fig. 1). To facilitate Region III SAR development, two virtual screening methods structure-based docking (gold),25, 26 and ligand-based, shape similarity matching (ROCS),27, 28 were employed to identify analogs of the structurally complex tetramethyl-piperidine moiety of 1. Synthesis and biological evaluation of the computationally designed analogs and resulting biological profiles were employed to investigate the effects of the structural modifications on CD4-gp120 binding and inhibition of viral entry.

Section snippets

Results and discussion

Prior to conducting virtual screening studies we synthesized and tested many Region I compounds (c.f. compounds in Madani et al.19 and Supplementary Tables 1–6) demonstrating that modification of the phenyl group in the base of the Phe 43 cavity was poorly tolerated. An optimal p-chloro, m-fluoro phenyl ring substitution pattern (1, Table 1) was necessary for enhanced binding in the base of the gp120 cavity.19 We thus sought to explore variations in the oxalamide linker of Region II. Several

Conclusions

The discovery that NBD analogs inhibit gp120-CD4 binding,19 mimic CD4-induced conformational changes in gp120,21 and inhibit HIV infection by inducing a short-lived activated state of the virus22 argued for further SAR explorations of the NBD chemotype. Ortho-substitution on the phenyl ring of 41 and changes to the oxalamide core of 1 led to analogs that failed to inhibit CD4-gp120 binding (Table 1 and Supplementary Tables 1–6). Further exploration and testing of shape-based analogs of 1 did

Small molecule modeling

Molecules were constructed in MOE (MOE Molecular Operating Environment Chemical Computing Group, version 2005.06 (Montreal, Canada) (http://www.chemcomp.com), ionized using MOE’s WashMDB function, and hydrogens were added.32 The small molecule conformation was minimized to a gradient of 0.01 in the MMFF94x33, 34 force field using a distance-dependent dielectric constant of 1.

Protein modeling

Using the X-ray crystal structure of the CD4-bound HIV-1 gp120 core15 (PDB code 1G9M), hydrogen atoms were added and

Acknowledgments

We thank Irwin Chaiken and Wayne Hendrickson and all the members of the PO1 Consortium Structure-Based Antagonism of HIV-1 Envelope Function in Cell Entry. Funding was provided by NIH GM 56550 to JL, EF, ABS, and JS. A. Sugawara thanks the Japan Society for the Promotion of Science for research fellowship support. JL thanks the Pittsburgh Supercomputing Center for an allocation for computing resources #MCB090108.

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