A p53 axis regulates B cell receptor-triggered, innate immune system-driven B cell clonal expansion.

Resting mature human B cells undergo a dynamic process of clonal expansion, followed by clonal contraction, during an in vitro response to surrogate C3d-coated Ag and innate immune system cytokines, IL-4 and BAFF. In this study, we explore the mechanism for clonal contraction through following the time- and division-influenced expression of several pro- and anti-apoptotic proteins within CFSE-labeled cultures. Several findings, involving both human and mouse B cells, show that a mitochondria-dependent apoptotic pathway involving p53 contributes to the high activation-induced cell death (AICD) susceptibility of replicating blasts. Activated B cell clones exhibit elevated p53 protein and elevated mRNA/protein of proapoptotic molecules known to be under direct p53 transcriptional control, Bax, Bad, Puma, Bid, and procaspase 6, accompanied by reduced anti-apoptotic Bcl-2. Under these conditions, Bim levels were not increased. The finding that full-length Bid protein significantly declines in AICD-susceptible replicating blasts, whereas Bid mRNA does not, suggests that Bid is actively cleaved to short-lived, proapoptotic truncated Bid. AICD was diminished, albeit not eliminated, by p53 small interfering RNA transfection, genetic deletion of p53, or Bcl-2 overexpression. DNA damage is a likely trigger for p53-dependent AICD because susceptible lymphoblasts expressed significantly elevated levels of both phosphorylated ataxia telangiectasia mutated-Ser(1980) and phospho-H2AX-Ser(139). Deficiency in activation-induced cytosine deaminase diminishes but does not ablate murine B cell AICD, indicating that activation-induced cytosine deaminase-induced DNA damage is only in part responsible. Evidence for p53-influenced AICD during this route of T cell-independent clonal expansion raises the possibility that progeny bearing p53 mutations might undergo positive selection in peripherally inflamed tissues with elevated levels of IL-4 and BAFF.

Disruption of estrogen receptor DNA-binding domain and related intramolecular communication restores tamoxifen sensitivity in resistant breast cancer.

A serious obstacle to successful treatment of estrogen receptor (ER)-positive human breast cancer is cell resistance to tamoxifen (TAM) therapy. Here we show that the electrophile disulfide benzamide (DIBA), an ER zinc finger inhibitor, blocks ligand-dependent and -independent cell growth of TAM-resistant breast cancer in vitro and in vivo. Such inhibition depends on targeting disruption of the ER DNA-binding domain and its communication with neighboring functional domains, facilitating ERalpha dissociation from its coactivator AIB1 and concomitant association with its corepressor NCoR bound to chromatin. DIBA does not affect phosphorylation of HER2, MAPK, AKT, and AIB1, suggesting that DIBA-modified ERalpha may induce a switch from agonistic to antagonistic effects of TAM on resistant breast cancer cells.

Small-molecule inactivation of HIV-1 NCp7 by repetitive intracellular acyl transfer.

The zinc fingers of the HIV-1 nucleocapsid protein, NCp7, are prime targets for antiretroviral therapeutics. Here we show that S-acyl-2-mercaptobenzamide thioester (SAMT) chemotypes inhibit HIV by modifying the NCp7 region of Gag in infected cells, thereby blocking Gag processing and reducing infectivity. The thiol produced by SAMT reaction with NCp7 is acetylated by cellular enzymes to regenerate active SAMTs via a recycling mechanism unique among small-molecule inhibitors of HIV.

Preclinical evaluation of a zinc finger inhibitor targeting lentivirus nucleocapsid protein in SIV-infected monkeys.

There is a continued need to develop inexpensive and effective drugs specific for novel targets of human immunodeficiency virus type 1 (HIV-1). The HIV-1 nucleocapsid p7 (NCp7) protein plays a critical role in early and late stages of the virus life cycle and possesses two highly conserved retroviral zinc fingers that are essential for its function. We have previously shown that zinc finger inhibitors (ZFI) based on the S-acyl 2-mercaptobenzamide thioester (SAMT) chemotype specifically target HIV NCp7 and are effective at reducing levels of infectious virus in an HIV-1-transgenic mouse model. Here, we did an initial proof-of-concept study to test the potential of a lead SAMT compound to reduce virus infectivity in the simian immunodeficiency virus (SIV) nonhuman primate model. SAMT-19 had potent antiviral and virucidal effects against the primary pathogenic isolate SIV/DeltaB670 and was non-cytotoxic in vitro. Cynomolgus macaques were infected intrarectally with SIV/DeltaB670 and treated with a low dose of SAMT-19 by continuous infusion from day 8 to day 28 post infection. Monkeys in the treatment group had significantly lower levels of infectious virus in peripheral blood mononuclear cells during the course of therapy as compared to monkeys in the control group, although therapy had no demonstrable effect on virus load. SAMT-19 therapy did not alter liver, kidney or immunologic function and was well tolerated by all treated monkeys. These data demonstrate that SAMT-19 is safe and virucidal in the nonhuman primate model. Further studies directed at optimizing SAMT bioavailability and pharmacokinetics likely will result in enhanced therapeutic efficacy of this promising HIV therapeutic.

Studies on the mechanism of inactivation of the HIV-1 nucleocapsid protein NCp7 with 2-mercaptobenzamide thioesters.

The HIV-1 nucleocapsid protein (NCp7) is a small basic protein with two CysCysHisCys zinc-binding domains that specifically recognizes the Psi-site of the viral RNA. NCp7 plays a number of crucial roles in the viral lifecycle, including reverse transcription and RNA encapsidation. Several classes of potential anti-HIV compounds have been designed to inactivate NCp7 through zinc ejection, including a special class of thioester compounds. We have investigated the mechanism of action of two N-substituted-S-acyl-2-mercaptobenzamide compounds (compounds 1 and 2) that target NCp7. UV/Visible spectroscopy studies demonstrated that both thioesters were able to eject metal from NCp7. NMR and mass spectroscopy studies showed that the thioester compounds specifically ejected zinc from the carboxyl-terminal zinc-binding domain of NCp7 by covalent modification of Cys(39). Exposure of NCp7 to compounds 1 and 2 destroyed its ability to specifically bind RNA, whereas NCp7 already bound to RNA was protected from zinc ejection by the thioesters. The thiol component of the thioesters (compound 3, 2-mercaptobenzoyl-beta-alaninamide) did not eject zinc from NCp7, but when compound 3 was incubated with acetyl CoA prior to incubation with NCp7, we observed extensive metal ejection. Thus, the thiol released by the reaction of compounds 1 and 2 could be re-acylated in vivo by acyl CoA to form a new thioester compound that is able to react with NCp7. These studies provide a better understanding of the mechanism of action of thioester compounds, which is important for future design of anti-HIV-1 compounds that target NCp7.

In vivo antiviral activity of novel human immunodeficiency virus type 1 nucleocapsid p7 zinc finger inhibitors in a transgenic murine model.

Control of human immunodeficiency virus through the use of inexpensive chemotherapeutics, with minimal side effects and decreased potential for engendering resistant virus, is a long-term therapeutic goal. In principle, this goal can be accomplished if viral replication in reservoirs of chronically and latently infected cells is addressed. As a first step, we have developed novel antiviral compounds based on a 2-mercaptobenzamide thioester chemotype, including the pyridinioalkanoyl thioesters, which specifically target the zinc fingers of the human immunodeficiency virus nucleocapsid protein (NCp7). Using these compounds in a murine transgenic model, in which infectious human immunodeficiency virus is induced from an integrated provirus, we show inhibition of transgenic spleen cell p24 expression with potencies comparable to acute infection assays using human peripheral blood lymphocytes. More importantly, transgenic mice treated in vivo with two 2-mercaptobenzamide thioesters expressed significantly lower plasma p24, and splenocytes from these animals produced fewer infectious virions. Thus, these thioesters may provide an effective means for inhibiting the expression of human immunodeficiency virus from integrated viral reservoirs.

Specificity of acyl transfer from 2-mercaptobenzamide thioesters to the HIV-1 nucleocapsid protein.

The HIV-1 nucleocapsid protein (NCp7) is a small, highly conserved protein with two zinc-binding domains that are essential for the protein's function. Molecules that bind to and inactivate NCp7 are currently being evaluated as new antiviral drugs. In particular, derivatives based on a 2-mercaptobenzamide thioester template have been shown to specifically eject zinc from the C-terminal zinc-binding domain (ZD2) of NCp7 via acyl transfer from the thioester to a cysteine sulfur. In this study, mutational analysis of the NCp7 amino acid sequence has been used to investigate the specificity of the interaction between ZD2 and a 2-mercaptobenzamide thioester compound using UV-vis spectroscopy and mass spectrometry to monitor the rate of metal ejection from NCp7 mutant peptides and sites of acylation, respectively. We were able to extend the previously reported mechanism of action of these thioester compounds to include a secondary S to N intramolecular acyl transfer that occurs after the primary acyl transfer from the thioester to a cysteine side chain in the protein. Structural models of the thioester/ZD2 complex were then examined to identify the most likely binding orientation. We determined that position x+1 (where x is Cys36) needs to be an aromatic residue for reactivity and a hydrogen-bond donor in position x+9 is important for optimal reactivity. A basic residue (lysine or arginine) is required at position x+2 for the correct fold, while a lysine residue is needed for reactivity involving S to N acyl transfer. We report highly specific interactions between 2-mercaptobenzamide thioester compounds and NCp7 that offer a structural basis for refining and designing new antiretroviral therapeutics, directed toward a target that is resistant to viral mutation.

APRIL and BAFF promote increased viability of replicating human B2 cells via mechanism involving cyclooxygenase 2.

Of relevance to both protective and pathogenic responses to Ag is the recent finding that soluble molecules of the innate immune system, i.e., IL-4, B cell-activation factor of the TNF family (BAFF), and C3, exhibit significant synergy in promoting the clonal expansion of human B2 cells following low-level BCR ligation. Although IL-4, BAFF, and C3dg each contribute to early cell cycle entry and progression to S phase, only BAFF promotes later sustained viability of progeny needed for continued cycling. The present study sought to further clarify the mechanisms for BAFF's multiple functions. By comparing BAFF and a proliferation-inducing ligand (APRIL) efficacy at different stages in the response (only BAFF binds BR3; both bind transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) and B cell maturation Ag, the early role was attributed to BR3, while the later role was attributed to TACI/B cell maturation Ag. Importantly, BAFF- and APRIL-promoted viability of cycling lymphoblasts was associated with sustained expression of cyclooxygenase 2 (COX-2), the rate-limiting enzyme for PGE2 synthesis, within replicating cells. Supernatants of cultures with BAFF and APRIL contained elevated PGE2. Although COX-2 inhibitors diminished daughter cell viability, exogenous PGE2 (1-1000 nM) increased the viability and recovery of lymphoblasts. Increased yield of viable progeny was associated with elevated Mcl-1, suggesting that a BAFF/APRIL --> TACI --> COX-2 --> PGE2--> Mcl-1 pathway reduces activation-related, mitochondrial apoptosis in replicating human B2 cell clones.

Comparison of the specificity of interaction of cellular and viral zinc-binding domains with 2-mercaptobenzamide thioesters.

The interactions of two 2-mercaptobenzamide thioester compounds with six diverse zinc-binding domains (ZBDs) have been analyzed by UV/visible spectroscopy, NMR spectroscopy, and nucleic acid binding assays. These thioester compounds serve as useful tools for probing the intrinsic chemical stability of ZBDs that exist within a variety of cellular and viral proteins. In our studies, the classical (Cys(2)His(2)) zinc finger ZBDs, the interleaved RING like ZBDs of protein kinase C delta (Cys(2)HisCys and HisCys(3)), and the carboxyl-terminal (Cys(2)HisCys) ZBD of Mouse Mammary Tumor Virus nucleocapsid protein (MMTV NCp10) were resistant to reaction with the thioester compounds. In contrast, the thioester compounds were able to efficiently eject zinc from the amino-terminal (Cys(2)HisCys) ZBD of MMTV NCp10, a Cys(2)HisCys ZBD from Friend of GATA-1 (FOG-1), and from both Cys(4) ZBDs of GATA-1. In all cases, zinc ejection led to a loss of protein structure. Interestingly, GATA-1 was resistant to reaction with the thioester compounds when bound to its target DNA sequence. The electronic and steric screening was calculated for select ZBDs to further explore their reactivity. Based on these results, it appears that both first and second zinc-coordination shell interactions within ZBDs, as well as nucleic acid binding, play important roles in determining the chemical stability and reactivity of ZBDs. These studies not only provide information regarding the relative reactivity of cysteine residues within structural ZBDs but also are crucial for the design of future therapeutic agents that selectively target ZBDs, such as those that occur in the HIV-1 nucleocapsid protein.

Innate immunity and human B cell clonal expansion: effects on the recirculating B2 subpopulation.

Foci of autoantigen-specific B lymphocytes in nonlymphoid tissues have been associated with development of autoimmune disease. To better understand the genesis of such ectopic lymphoid tissue, this study investigated whether several B cell-tropic innate immune system molecules, known to be elevated in response to inflammatory stimuli, can cooperate in fostering the T cell-independent clonal expansion of mature human B2 cells under conditions of limiting BCR engagement. Notable synergy was observed between BCR coligation with the C3dg-binding CD21/CD19 costimulatory complex, B cell-activating factor belonging to the TNF family (BAFF), and IL-4 in generating B cell progeny with sustained CD86 and DR expression. The synergy was observed over a wide range of BCR:ligand affinities and involved: 1) cooperative effects at promoting early cell cycle progression and viability; 2) BCR:CD21 coligation-promoted increases in BAFF receptors that were highly regulated by IL-4; 3) reciprocal effects of IL-4 and BAFF at dampening daughter cell apoptosis typical of stimulation by BCR:CD21 and either cytokine alone; and 4) BAFF-sustained expression of antiapoptotic Mcl-1 within replicating lymphoblasts. The results suggest that significant clonal proliferation of recirculating B2 cells occurs upon limited binding to C3dg-coated Ag in an inflammatory in vivo milieu containing both BAFF and IL-4. When rare autoantigen-presenting B cells undergo such expansions, both B cell and T cell autoimmunity may be promoted.

Antigen receptor triggered upregulation of CD86 and CD80 in human B cells: augmenting role of the CD21/CD19 co-stimulatory complex and IL-4.

The impact of BCR:CD21 co-engagement on B cell expression of molecules critical for T cell activation was investigated with receptor-specific mAbs conjugated to high MW dextran as stimulatory ligands. In the absence of IL-4, BCR:CD21 co-ligation augmented BCR-triggered CD86 only under conditions of very low BCR ligand dose or affinity, and CD80 was minimally induced by BCR and/or CD21 crosslinking. In the presence of IL-4, BCR:CD21 co-ligation augmented CD86 and CD80 expression under conditions of greater BCR engagement. However, with very high level BCR engagement, no bonus effect of BCR:CD21 crosslinking was observed. Co-ligation-promoted CD86 and CD80 expression was associated with heightened B cell activation of resting allogeneic T cells. The data suggest that co-clustering of BCR and the CD21/CD19 co-stimulatory complex following B cell engagement with C3d-bound microbial or self-antigens will enhance B cell recruitment of T cell help only when IL-4 is present and/or BCR engagement is very limiting.

Role of complement-binding CD21/CD19/CD81 in enhancing human B cell protection from Fas-mediated apoptosis.

Defective expression of Fas leads to B cell autoimmunity, indicating the importance of this apoptotic pathway in eliminating autoreactive B cells. However, B cells with anti-self specificities occasionally escape such regulation in individuals with intact Fas, suggesting ways of precluding this apoptosis. Here, we examine whether coligation of the B cell Ag receptor (BCR) with the complement (C3)-binding CD21/CD19/CD81 costimulatory complex can enhance the escape of human B cells from Fas-induced death. This was warranted given that BCR-initiated signals induce resistance to Fas apoptosis, some (albeit not all) BCR-triggered events are amplified by coligation of BCR and the co-stimulatory complex, and several self Ags targeted in autoimmune diseases effectively activate complement. Using a set of affinity-diverse surrogate Ags (receptor-specific mAb:dextran conjugates) with varying capacity to engage CD21, it was established that BCR:CD21 coligation lowers the BCR engagement necessary for inducing protection from Fas apoptosis. Enhanced protection was associated with altered expression of several molecules known to regulate Fas apoptosis, suggesting a unique molecular model for how BCR:CD21 coligation augments protection. BCR:CD21 coligation impairs the generation of active fragments of caspase-8 via dampened expression of membrane Fas and augmented expression of FLIP(L). This, in turn, diminishes the generation of cells that would be directly triggered to apoptosis via caspase-8 cleavage of caspase 3 (type I cells). Any attempt to use the mitochondrial apoptotic protease-activating factor 1 (Apaf-1)-dependent pathway for apoptosis (as type II cells) is further blocked because BCR:CD21 coligation promotes up-regulation of the mitochondrial antiapoptotic molecule, Bcl-2.

Optimization of unique, uncharged thioesters as inhibitors of HIV replication.

A combinatorial chemistry approach was employed to prepare a restricted library of N-substituted S-acyl-2-mercaptobenzamide thioesters. It was shown that many members of this chemotype display anti-HIV activity via their ability to interact with HIV-1, HIV-2, SIV-infected cells, cell-free virus, and chronically and latently infected cells in a manner consistent with targeting of the highly conserved HIV-1 NCp7 zinc fingers. Compounds were initially screened using two different in vitro antiviral assays and evaluated for stability in neutral buffer containing 10% pooled human serum using a spectrophotometric assay. These data revealed that there was no significant correlation between thioester stability and antiviral activity, however, a slight inverse correlation between serum stability and virucidal activity was noted. Based on the virucidal capability and the ability to select lead compounds to inhibit virus expression from latently infected TNFalpha-induced U1 cells, we next determined if these compounds could prevent HIV cell-to-cell transmission. Several thioesters demonstrated potent inhibition of HIV cell-to-cell transmission with EC50 values in the 80-100 nM range. Thus, we have optimized a series of restricted thioesters and provided evidence that serum stability is not required for antiviral activity. Moreover, selected compounds show potential for development as topical microbicides.

Synthesis and biological properties of amino acid amide ligand-based pyridinioalkanoyl thioesters as anti-HIV agents.

Hyper-mutable retroviruses such as HIV can become rapidly resistant to drugs used to treat infection. Strategies for coping with drug-resistant strains of virus include combination therapies, using viral protease and reverse transcriptase inhibitors. Another approach is the development of antiviral agents that attack mutationally nonpermissive targets that have functions essential for viral replication. Thus, the highly conserved nucleocapsid protein, NCp7, was chosen as a prime target in our search for novel anti-HIV agents that can overcome the problem of viral drug resistance. Recently, we reported (J. Med. Chem. 1999, 42, 67) a novel chemotype, the pyridinioalkanoyl thioesters (PATEs), based on 2-mercaptobenzamides as the thiol component and having its amide nitrogen substituted with various phenylsulfonyl moieties. These compounds were identified as relatively nontoxic anti-HIV agents in the XTT cytoprotection assay. In this study, we wish to report a separate genre of active PATEs wherein the thiol component consists of an N-2-mercaptobenzoyl-amino acid derivative. Active derivatives (EC(50) < 10 microM) reported herein were confined to amino acid primary amides or methyl amides having side chains no larger than isobutyl. Amino acids terminating in free carboxyl or carboxylic acid ester groups were mostly inactive. Selected compounds were shown to be active on chronically infected CEM/SK-1, TNFalpha-induced U1, ACH-2 cells and virucidal on cell-free virus, latently infected U1 cells and acutely infected primary peripheral blood mononuclear cells (PBMCs).

Benzamide-based thiolcarbamates: a new class of HIV-1 NCp7 inhibitors.

The HIV-1 nucleocapsid protein NCp7, which contains two highly conserved zinc fingers, is being used as a novel target for AIDS therapy due to its pivotal role in viral replication and its mutationally intolerant nature. Herein we report a new class of NCp7 inhibitors that possess good antiviral activity with low cellular toxicity.