Current Pharmaceutical Design - Volume 10, Issue 15, 2004

This review focuses on our approach to the study of the effect of a series of phosphoramidate substituted nucleoside analogs on model systems for cancer, HIV and fertility. This approach allowed the development of compound WHI-07, an arylphosphoramidate derivative of zidavudine. This compound is a multifunctional agent showing potent activity in the above mentioned model systems. Our rational drug design provided such a powerful derivative with all the necessary characteristic of a drug candidate. Importantly, we have experimental evidence that each of the groups associated with the molecular frame of WHI-07 imparts the multifunctional ability for this agent. In addition, we have also suggested a possible biological pathway for WHI-07 including various products with their therapeutic targets that are formed during the course of its metabolism inside the cell. We also propose which individual moieties in the structure of WHI-07 are responsible for the biological activity from the formation of these metabolites. A detailed structure-activity relationship is presented in the review in connection with various structural modifications of the agent. Application of this active agent in animal models shows the potential usefulness of this agent as a drug candidate. We further plan to utilize gene-chip technology to identify new targets and modes of action using microarrays to measure expression changes in thousands of gene products. In conclusion, we have demonstrated the power of multifunctional drug design to discover drugs to combat various diseases. We believe this is the future direction of the drug discovery process.

Mast cells and basophils are major effector cells in the immunoglobulin E (IgE)-dependent allergic reactions as well as in the innate immunity. They are distributed throughout the body and, upon allergen exposure, are stimulated via the high affinity IgE receptor (FcεRI) to release several pro-inflammatory mediators such as leukotrienes, immunoregulatory cytokines and histamine. FcεRI-mediated signaling is initiated by tyrosine phosphorylation of FcεRI subunits by Src family kinase Lyn, which is followed by an activation of Syk / Zap family kinase Syk. The activated kinases then in turn phosphorylate and activate other enzymes [phospholipase Cγ (PLCγ) isoforms, phosphatidylinositol-3 kinase (PI3K) isoforms, protein kinase C (PKC) isoforms, Bruton's tyrosine kinase (Btk) and others], adaptors [linker for activation of T cells (LAT), Cbl, Grb2 and others] and GTP exchange factors / GTPases (Vav, Ras, Rho, and others), and subsequently induce the mobilization of stored and extracellular Ca2+. These and other biochemical events lead within seconds and minutes to the secretory response and later to the production of chemokines. This review is focused on the use of tyrosine kinase inhibitors specific for Src family kinases (PP1 / PP2, SU6656 and CT5269), Syk kinase (piceatannol, ER-27319 and BAY 61-3606) and Btk (terreic acid and LFM-A13) for a modulation of FcεRI-mediated signaling in mast cells. Potential use of the inhibitors in the treatment of inflammatory and allergy diseases as well as future directions in the development of highly specific tyrosine kinases inhibitors of new generations and their use in an intended modulation of mast cell signaling are discussed.

The spleen tyrosine kinase Syk is an enigmatic protein tyrosine kinase functional in a number of diverse cellular processes. It is best known as a non receptor protein tyrosine kinase involved in signal transduction in cells of hematopoietic origin and plays a crucial role in signaling in most of these cells. It is involved in B and T-cell function, platelet aggregation, mast cell signaling, neutrophils and macrophages. Recently it has been found in tissues outside of the hematopoietic lineage. Perhaps the most interesting non-traditional role of Syk is that of a potential tumor suppressor in breast cancer. Absence of Syk protein in primary breast tumors is correlated with poor outcomes. Syk deficient cells have increased motility which is restored to normalcy by replacement with wild-type Syk. Syk also associates with the actin and tubulin cytoskeleton and is an α-tubulin kinase. The central role that Syk has in a number of cellular processes makes it an ideal starting point for broad therapeutic targeting.

The cellular signaling machinery is a complex network of cross-talking proteins that enables dynamic communication between upstream causal factors and downstream effectors. Non-receptor tyrosine kinases, including Src, are the intermediates of signal transfer, controlling pathways as diverse as cell growth, death, differentiation, migration, and genome maintenance. When expressed as viral genes these proteins are potent carcinogens. Furthermore, analogous genetic alterations are observed, albeit not frequently, in human tumors. In a variety of tumors including those derived from the colon and breast, Src is either over expressed or constitutively active in a large percentage of patients. Increased expression or activity of Src correlates with the stage and metastatic potential of some neoplasia. The detailed knowledge of Src activation facilitates rational design of drugs that potentially interfere with either binding of ATP or substrate peptides. Several existing inhibitors are available as lead compounds for further development of Src inhibitors.

BTK (Bruton's tyrosine kinase) is a member of the TEC family of tyrosine kinases that plays a central but diverse modulatory role in various cellular processes. The unique role of BTK in a multitude of signaling pathways, its function as a dual regulator of apoptosis and its involvement in a number of developmental processes makes BTK a desirable target for potential anti-cancer, anti-inflammatory and anti-viral agents as well as other treatments. The biochemistry and signaling networks of BTK were well described in numerous detailed reviews written by members of our team and others before us. Therefore in this particular review we are going to concentrate on the possible practical application of previously obtained knowledge to specific diseases and disorders.

Here we discuss the therapeutic potential of Janus kinase 3 (JAK3) inhibitors as a new class of immunomodulatory agents with immunosuppressive, anti-inflammatory, anti-allergic, anti-thrombotic and anti-leukemic properties. JAKs are abundantly expressed in primary leukemic cells from children with ALL (acute lymphoblastic leukemia) and are crucial for signals regulating apoptosis. Additional roles for JAK3 in mast cell-mediated immediate hypersensitivity reactions, autoimmune disorders and platelet function have recently been described. The preclinical studies on JAK3 inhibitors revealed their clinical potential as anti-leukemic agents with anti-thrombotic, anti-allergic and immunosuppressive properties. Results from multiple preclinical experimental model systems of autoimmune diabetes, pancreatic islet transplantation, solid organ transplantation, allergy, thrombosis and bone marrow transplantation are discussed in the context of the clinical need for new immunomodulatory agents with such properties.

BMS-378806 is a prototype of a new class of small molecule HIV-1 inhibitors that blocks viral attachment to cells. This compound exhibits potent inhibitory activity against a panel of HIV-1 laboratory and clinical isolates (M- and T-tropic), selective for HIV-1 and inactive against HIV-2, SIV and a panel of other viruses. BMS-378806 exhibits no significant cytotoxicity and displays many attractive pharmacological properties such as low protein binding, minimal serum effect on anti-HIV-1 potency, good oral bioavailability in animal species and a clean safety profile in initial animal toxicology studies. The compound binds to gp120 and blocks the attachment of the HIV-1 envelope protein to cellular CD4 receptors via a specific and competitive mechanism. BMS-378806 binds directly to gp120 at an approximately 1:1 stoichiometry, with a binding affinity similar to that of soluble CD4. Further confirmation that this class of compounds targets the envelope in infected cells was obtained through the isolation of resistant variants and the mapping of resistance substitutions to the HIV-1 envelope. In particular, two substitutions, M426L and M475I, are situated at or near the CD4 binding pocket of gp120. Recombinant HIV-1 carrying these two substitutions demonstrated significantly reduced susceptibility to inhibition. Using these HIV-1 gp120 resistant variants and gp120 / CD4 contact site mutants, the potential BMS-378806 target site was localized to a specific region within the CD4 binding pocket of gp120. Together, the data show that BMS-378806 is the first of a new class of HIV inhibitors with the potential to become a valued addition to our current repertoire of antiretroviral drugs.

The use of HAART with double or triple drug combinations has significantly improved the survival of AIDS patients. However, the emergence of virus-drug resistance and both short- and long-term drug-related side effects are among the main reasons for continuing the development of new classes of effective anti-HIV drugs that target the replicative cycle at different sites. In recent years, tremendous progress has been made in understanding HIV-1 entry, a multistep process that comprises viral attachment, coreceptor interactions and fusion. The mechanistic insight gained from these studies has enabled the design of specific agents that can inhibit each step in the HIV entry process. The successful results from clinical trials with enfuvirtide (T-20), the first approved entry inhibitor, indicate that targeting of HIV entry will soon be an important component of antiretroviral therapy and further encourage the development of effective entry inhibitors. In this article the recent developments of therapeutic agents endowed with inhibitory properties against the binding of the HIV envelope glycoprotein gp120 to the CD4 receptor (e.g., PRO 542, BMS-378806, TNX-355, PRO 2000 and CV-N) are briefly outlined. Major focus is placed on the anti-HIV activity of cyclotriazadisulfonamides (CADA), a novel class of compounds with a unique mode of action by down-modulating the CD4 receptor in lymphocytic and monocytic cells.

The recent success of the fusion inhibitor T-20 (enfuvirtide) in clinical studies has ushered in a new chapter in the development of anti-HIV-1 therapeutics. T-20 is the first FDA-approved drug that targets the viral transmembrane protein gp41. This protein, along with gp120, promotes viral entry through a coordinated cascade of conformational transitions that lead to the fusion of the HIV-1 and target cell membranes. The interaction of gp120 with CD4 and a chemokine receptor stimulates gp41 to extend and bridge the space between the virus and cell. Subsequently, gp41 collapses into a trimer-of-hairpins structure that brings the viral and cellular membranes into close proximity necessary for fusion. Enfuvirtide targets the gp41 amino-terminal region exposed in the transient extended state, blocking the ultimate collapse into the trimer-of hairpins and inhibiting membrane fusion. The vulnerability of this transient extended state has stimulated the development of new agents, ranging from small molecules to large proteins, that bind to gp41 and inhibit its structural transformations. The discovery and characterization of these inhibitors have not only led to new antiviral strategies, but have also shed light on the accessibility of gp41 epitopes that might play a role in HIV-1 vaccine development.

The gp41 subunit of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein plays an important role in HIV-1 entry and severs as an attractive target for development of HIV-1 entry inhibitors, a new class of anti-HIV drugs. Triggered by gp120 binding to CD4 and a coreceptor, gp41 undergoes a conformation shift from a native prefusogenic state to a fusogenic state, in which the N-terminal heptad repeat (NHR) and C-terminal heptad repeat (CHR) associate to form a six-helix bundle, representing the fusion-active gp41 core. Any compound that disrupts the gp41 sixhelix bundle formation may inhibit the gp41-mediated membrane fusion, thereby blocking HIV-1 entry into target cells. Peptides derived from the gp41 NHR and CHR regions, designated N- and C-peptides, can interact with the counterpart regions in gp41 and interfere with the six-helix bundle formation between the viral NHR and CHR region, thus inhibiting fusion of the virus with the target cell. One of the C-peptides, T-20 (brand name: Fuzeon), was recently approved by the US FDA as the first HIV entry inhibitor which can be used for treatment of AIDS patients who fail to respond to the current antiretroviral drugs, e.g., the reverse transcriptase inhibitors and protease inhibitors. The limitations of T-20 include lack of oral availability and high cost of production. Thus it is essential to develop small molecule HIV-1 entry inhibitors targeting gp41. This review summarizes the newly developed techniques for high throughput screening (HTS) and characterization of the HIV-1 entry inhibitors targeting gp41. The theories behind these techniques are also discussed. It is expected that the “drug-like” compounds with potent HIV-1 fusion inhibitory activity will be identified in the near future and used as leads for development of the low molecular weight HIV-1 entry inhibitors for the chemotherapy of HIV-1 infection and AIDS.

An increasing number of human immunodeficiency virus (HIV)-infected patients have detectable viraemia despite treatment with multiple-drug combinations or have developed resistance to all available classes of antiretroviral therapy. HIV entry has become an important pharmacological target. Enfuvirtide is the first HIV entry inhibitor to be approved for the treatment of drug-experienced patients but several other agents are progressing through preclinical and clinical trials. However, because different entry inhibitors target different parts of the entry process their combined effects could be synergistic or generate different distinct profiles of drug-resistance. The HIV envelope glycoprotein that drives HIV entry is highly variable. Its plasticity allows HIV to escape the immune system and its variability is associated with HIV tropism, fitness and replicative capacity. Thus, mutations that confer resistance to entry inhibitors will modify these parameters. Therapeutic strategies that aim at blocking virus entry may also be used to alter the natural evolution of HIV in an unprecedented way. Here, we will describe the structure and function of the envelope glycoprotein complex that constitute the basis for the emergence of resistance to HIV entry inhibitors, review those HIV entry inhibitors for which drug-resistance has been evaluated and discuss the interplay between viral resistance to inhibitors of HIV entry and the pathogenicity of HIV and AIDS.

Inhibiting the HIV-1 entry process offer a new therapeutic target and the hope to potentialize our current treatments against wild-type or drug-resistant viruses. Several inhibitors of CD4, co-receptor CCR5 or CXCR4 and fusion are at various levels of clinical development. How best to use this class of drugs in our therapeutic arsenal remains to be defined. It is likely that these compounds will not be used as monotherapy. Therefore, it is important to evaluate how these drugs will interact among themselves as well as with antiretrovirals from other classes. Drug interactions can range from synergy to antagonism depending on factors including binding affinity, drug concentrations, and pharmacokinetics. In the case of entry inhibitors, one must also consider that the entry of HIV-1 into the cell is a multi-step process that involve cumulative events which are interdependent. Furthermore, polymorphism both in the coreceptors and in gp120, the density of coreceptors, and the binding site of the drug may also affect efficacy. Therefore it is difficult to predict how blocking one step of the process will affect the subsequent one without carefully studying interactions of each potential combination in an in vitro system. So far, studies of interactions between fusion inhibitors and coreceptor inhibitors have shown a high level of synergy. Similar studies performed with two co-receptor inhibitors have shown results varying from synergy to high antagonism depending on the viral isolate and the compounds used. In the following chapter, we will review some concepts of mechanisms that may affect these interactions.