Judith M. LaLonde

Indoleamine 2,3-Dioxygenase Is the Anticancer Target for a Novel Series of Potent Naphthoquinone-Based Inhibitors

Indoleamine 2,3-dioxygenase (IDO) is emerging as an important new therapeutic target for the treatment of cancer, chronic viral infections, and other diseases characterized by pathological immune suppression. While small molecule inhibitors of IDO exist, there remains a dearth of high-potency compounds offering in vivo efficacy and clinical translational potential. In this study, we address this gap by defining a new class of naphthoquinone-based IDO inhibitors exemplified by the natural product menadione, which is shown in mouse tumor models to have similar antitumor activity to previously characterized IDO inhibitors. Genetic validation that IDO is the critical in vivo target is demonstrated using IDO-null mice. Elaboration of menadione to a pyranonaphthoquinone has yielded low nanomolar potency inhibitors, including new compounds which are the most potent reported to date (K(i) = 61-70 nM). Synthetic accessibility of this class will facilitate preclinical chemical-genetic studies as well as further optimization of pharmacological parameters for clinical translation.

Small-Molecule CD4 Mimics Interact with a Highly Conserved Pocket on HIV-1 gp120

Human immunodeficiency virus (HIV-1) interaction with the primary receptor, CD4, induces conformational changes in the viral envelope glycoproteins that allow binding to the CCR5 second receptor and virus entry into the host cell. The small molecule NBD-556 mimics CD4 by binding the gp120 exterior envelope glycoprotein, moderately inhibiting virus entry into CD4-expressing target cells and enhancing CCR5 binding and virus entry into CCR5-expressing cells lacking CD4. Studies of NBD-556 analogs and gp120 mutants suggest that (1) NBD-556 binds within the Phe 43 cavity, a highly conserved, functionally important pocket formed as gp120 assumes the CD4-bound conformation; (2) the NBD-556 phenyl ring projects into the Phe 43 cavity; (3) enhancement of CD4-independent infection by NBD-556 requires the induction of conformational changes in gp120; and (4) increased affinity of NBD-556 analogs for gp120 improves antiviral potency during infection of CD4-expressing cells.

Localized Changes in the gp120 Envelope Glycoprotein Confer Resistance to Human Immunodeficiency Virus Entry Inhibitors BMS-806 and #155

ABSTRACT BMS-806 and the related compound, #155, are novel inhibitors of human immunodeficiency virus type 1 (HIV-1) entry that bind the gp120 exterior envelope glycoprotein. BMS-806 and #155 block conformational changes in the HIV-1 envelope glycoproteins that are induced by binding to the host cell receptor, CD4. We tested a panel of HIV-1 envelope glycoprotein mutants and identified several that were resistant to the antiviral effects of BMS-806 and #155. In the CD4-bound conformation of gp120, the amino acid residues implicated in BMS-806 and #155 resistance line the “phenylalanine 43 cavity” and a water-filled channel that extends from this cavity to the inner domain. Structural considerations suggest a model in which BMS-806 and #155 bind gp120 prior to receptor binding and, upon CD4 binding, are accommodated in the Phe-43 cavity and adjacent channel. The integrity of the nearby V1/V2 variable loops and N-linked carbohydrates on the V1/V2 stem indirectly influences sensitivity to the drugs. A putative binding site for BMS-806 and #155 between the gp120 receptor-binding regions and the inner domain, which is thought to interact with the gp41 transmembrane envelope glycoprotein, helps to explain the mode of action of these drugs.

Structure Based Development of Phenylimidazole-Derived Inhibitors of Indoleamine 2,3-Dioxygenase

Indoleamine 2,3-dioxygenase (IDO) is emerging as an important new therapeutic target for the treatment of cancer, chronic viral infections, and other diseases characterized by pathological immune suppression. With the goal of developing more potent IDO inhibitors, a systematic study of 4-phenylimidazole (4-PI) derivatives was undertaken. Computational docking experiments guided design and synthesis efforts with analogues of 4-PI. In particular, three interactions of 4-PI analogues with IDO were studied: the active site entrance, the interior of the active site, and the heme iron binding. The three most potent inhibitors (1, 17, and 18) appear to exploit interactions with C129 and S167 in the interior of the active site. All three inhibitors are approximately 10-fold more potent than 4-PI. The study represents the first example of enzyme inhibitor development with the recently reported crystal structure of IDO and offers important lessons in the search for more potent inhibitors.

Validation Studies of the Site-Directed Docking Program LibDock

The performance of the site-features docking algorithm LibDock has been evaluated across eight GlaxoSmithKline targets as a follow-up to a broad validation study of docking and scoring software (Warren, G. L.; Andrews, W. C.; Capelli, A.; Clarke, B.; Lalonde, J.; Lambert, M. H.; Lindvall, M.; Nevins, N.; Semus, S. F.; Senger, S.; Tedesco, G.; Walls, I. D.; Woolven, J. M.; Peishoff, C. E.; Head, M. S. J. Med. Chem. 2006, 49, 5912-5931). Docking experiments were performed to assess both the accuracy in reproducing the binding mode of the ligand and the retrieval of active compounds in a virtual screening protocol using both the DJD (Diller, D. J.; Merz, K. M., Jr. Proteins 2001, 43, 113-124) and LigScore2 (Krammer, A. K.; Kirchoff, P. D.; Jiang, X.; Venkatachalam, C. M.; Waldman, M. J. Mol. Graphics Modell. 2005, 23, 395-407) scoring functions. This study was conducted using DJD scoring, and poses were rescored using all available scoring functions in the Accelrys LigandFit module, including LigScore2. For six out of eight targets at least 30% of the ligands were docked within a root-mean-square difference (RMSD) of 2.0 A for the crystallographic poses when the LigScore2 scoring function was used. LibDock retrieved at least 20% of active compounds in the top 10% of screened ligands for four of the eight targets in the virtual screening protocol. In both studies the LigScore2 scoring function enhanced the retrieval of crystallographic poses or active compounds in comparison with the results obtained using the DJD scoring function. The results for LibDock accuracy and ligand retrieval in virtual screening are compared to 10 other docking and scoring programs. These studies demonstrate the utility of the LigScore2 scoring function and that LibDock as a feature directed docking method performs as well as docking programs that use genetic/growing and Monte Carlo driven algorithms.

Structure-Based Design, Synthesis, and Characterization of Dual Hotspot Small-Molecule HIV-1 Entry Inhibitors.

Cellular infection by HIV-1 is initiated with a binding event between the viral envelope glycoprotein gp120 and the cellular receptor protein CD4. The CD4-gp120 interface is dominated by two hotspots: a hydrophobic gp120 cavity capped by Phe43(CD4) and an electrostatic interaction between residues Arg59(CD4) and Asp368(gp120). The CD4 mimetic small-molecule NBD-556 (1) binds within the gp120 cavity; however, 1 and related congeners demonstrate limited viral neutralization breadth. Herein, we report the design, synthesis, characterization, and X-ray structures of gp120 in complex with small molecules that simultaneously engage both binding hotspots. The compounds specifically inhibit viral infection of 42 tier 2 clades B and C viruses and are shown to be antagonists of entry into CD4-negative cells. Dual hotspot design thus provides both a means to enhance neutralization potency of HIV-1 entry inhibitors and a novel structural paradigm for inhibiting the CD4-gp120 protein-protein interaction.

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

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.

Structure-based design, synthesis and validation of CD4-mimetic small molecule inhibitors of HIV-1 entry: conversion of a viral entry agonist to an antagonist.

Conspectus This Account provides an overview of a multidisciplinary consortium focused on structure-based strategies to devise small molecule antagonists of HIV-1 entry into human T-cells, which if successful would hold considerable promise for the development of prophylactic modalities to prevent HIV transmission and thereby alter the course of the AIDS pandemic. Entry of the human immunodeficiency virus (HIV) into target T-cells entails an interaction between CD4 on the host T-cell and gp120, a component of the trimeric envelope glycoprotein spike on the virion surface. The resultant interaction initiates a series of conformational changes within the envelope spike that permits binding to a chemokine receptor, formation of the gp41 fusion complex, and cell entry. A hydrophobic cavity at the CD4–gp120 interface, defined by X-ray crystallography, provided an initial site for small molecule antagonist design. This site however has evolved to facilitate viral entry. As such, the binding of prospective small molecule inhibitors within this gp120 cavity can inadvertently trigger an allosteric entry signal. Structural characterization of the CD4–gp120 interface, which provided the foundation for small molecule structure-based inhibitor design, will be presented first. An integrated approach combining biochemical, virological, structural, computational, and synthetic studies, along with a detailed analysis of ligand binding energetics, revealed that modestly active small molecule inhibitors of HIV entry can also promote viral entry into cells lacking the CD4 receptor protein; these competitive inhibitors were termed small molecule CD4 mimetics. Related congeners were subsequently identified with both improved binding affinity and more potent viral entry inhibition. Further assessment of the affinity-enhanced small molecule CD4 mimetics demonstrated that premature initiation of conformational change within the viral envelope spike, prior to cell encounter, can lead to irreversible deactivation of viral entry machinery. Related congeners, which bind the same gp120 site, possess different propensities to elicit the allosteric response that underlies the undesired enhancement of CD4-independent viral entry. Subsequently, key hotspots in the CD4–gp120 interface were categorized using mutagenesis and isothermal titration calorimetry according to the capacity to increase binding affinity without triggering the allosteric signal. This analysis, combined with cocrystal structures of small molecule viral entry agonists with gp120, led to the development of fully functional antagonists of HIV-1 entry. Additional structure-based design exploiting two hotspots followed by synthesis has now yielded low micromolar inhibitors of viral entry.

Structural Determinants for Affinity Enhancement of a Dual Antagonist Peptide Entry Inhibitor of Human Immunodeficiency Virus Type-1

Structure-activity correlations were investigated for substituted peptide conjugates that function as dual receptor site antagonists of HIV-1 gp120. A series of peptide conjugates were constructed via click reaction of both aryl and alkyl acetylenes with an internally incorporated azidoproline 6 derived from the parent peptide 1 (12p1, RINNIPWSEAMM). Compared to 1, many of these conjugates were found to exhibit several orders of magnitude increase in both affinity for HIV-1 gp120 and inhibition potencies at both the CD4 and coreceptor binding sites of gp120. We sought to determine structural factors in the added triazole grouping responsible for the increased binding affinity and antiviral activity of the dual inhibitor conjugates. We measured peptide conjugate potencies in both kinetic and cell infection assays. High affinity was sterically specific, being exhibited by the cis- but not the trans-triazole. The results demonstrate that aromatic, hydrophobic, and steric features in the residue 6 side-chain are important for increased affinity and inhibition. Optimizing these features provides a basis for developing gp120 dual inhibitors into peptidomimetic and increasingly smaller molecular weight entry antagonist leads.

CD4-Mimetic Small Molecules Sensitize Human Immunodeficiency Virus to Vaccine-Elicited Antibodies

ABSTRACT Approaches to prevent human immunodeficiency virus (HIV-1) transmission are urgently needed. Difficulties in eliciting antibodies that bind conserved epitopes exposed on the unliganded conformation of the HIV-1 envelope glycoprotein (Env) trimer represent barriers to vaccine development. During HIV-1 entry, binding of the gp120 Env to the initial receptor, CD4, triggers conformational changes in Env that result in the formation and exposure of the highly conserved gp120 site for interaction with the coreceptors, CCR5 and CXCR4. The DMJ compounds (+)-DMJ-I-228 and (+)-DMJ-II-121 bind gp120 within the conserved Phe 43 cavity near the CD4-binding site, block CD4 binding, and inhibit HIV-1 infection. Here we show that the DMJ compounds sensitize primary HIV-1, including transmitted/founder viruses, to neutralization by monoclonal antibodies directed against CD4-induced (CD4i) epitopes and the V3 region, two gp120 elements involved in coreceptor binding. Importantly, the DMJ compounds rendered primary HIV-1 sensitive to neutralization by antisera elicited by immunization of rabbits with HIV-1 gp120 cores engineered to assume the CD4-bound state. Thus, small molecules like the DMJ compounds may be useful as microbicides to inhibit HIV-1 infection directly and to sensitize primary HIV-1 to neutralization by readily elicited antibodies. IMPORTANCE Preventing HIV-1 transmission is a priority for global health. Eliciting antibodies that can neutralize many different strains of HIV-1 is difficult, creating problems for the development of a vaccine. We found that certain small-molecule compounds can sensitize HIV-1 to particular antibodies. These antibodies can be elicited in rabbits. These results suggest an approach to prevent HIV-1 sexual transmission in which a virus-sensitizing microbicide is combined with a vaccine.