Fragment-based and structure-guided discovery of perforin inhibitors.

Perforin is a pore-forming protein whose normal function enables cytotoxic T and natural killer (NK) cells to kill virus-infected and transformed cells. Conversely, unwanted perforin activity can also result in auto-immune attack, graft rejection and aberrant responses to pathogens. Perforin is critical for the function of the granule exocytosis cell death pathway and is therefore a target for drug development. In this study, by screening a fragment library using NMR and surface plasmon resonance, we identified 4,4-diaminodiphenyl sulfone (dapsone) as a perforin ligand. We also found that dapsone has modest (mM) inhibitory activity of perforin lytic activity in a red blood cell lysis assay in vitro. Sequential modification of this lead fragment, guided by structural knowledge of the ligand binding site and binding pose, and supported by SPR and ligand-detected 19F NMR, enabled the design of nanomolar inhibitors of the cytolytic activity of intact NK cells against various tumour cell targets. Interestingly, the ligands we developed were largely inert with respect to direct perforin-mediated red blood cell lysis but were very potent in the context of perforin's action on delivering granzymes in the immune synapse, the context in which it functions physiologically. Our work indicates that a fragment-based, structure-guided drug discovery strategy can be used to identify novel ligands that bind perforin. Moreover, these molecules have superior physicochemical properties and solubility compared to previous generations of perforin ligands.

Small Molecule Inhibitors of Lymphocyte Perforin as Focused Immunosuppressants for Infection and Autoimmunity.

New drugs that precisely target the immune mechanisms critical for cytotoxic T lymphocyte (CTL) and natural killer (NK) cell driven pathologies are desperately needed. In this perspective, we explore the cytolytic protein perforin as a target for therapeutic intervention. Perforin plays an indispensable role in CTL/NK killing and controls a range of immune pathologies, while being encoded by a single copy gene with no redundancy of function. An immunosuppressant targeting this protein would provide the first-ever therapy focused specifically on one of the principal cell death pathways contributing to allotransplant rejection and underpinning multiple autoimmune and postinfectious diseases. No drugs that selectively block perforin-dependent cell death are currently in clinical use, so this perspective will review published novel small molecule inhibitors, concluding with in vivo proof-of-concept experiments performed in mouse models of perforin-mediated immune pathologies that provide a potential pathway toward a clinically useful therapeutic agent.

Preclinical Activity and Pharmacokinetic/Pharmacodynamic Relationship for a Series of Novel Benzenesulfonamide Perforin Inhibitors.

Perforin is a key effector of lymphocyte-mediated cell death pathways and contributes to transplant rejection of immunologically mismatched grafts. We have developed a novel series of benzenesulfonamide (BZS) inhibitors of perforin that can mitigate graft rejection during allogeneic bone marrow/stem cell transplantation. Eight such perforin inhibitors were tested for their murine pharmacokinetics, plasma protein binding, and their ability to block perforin-mediated lysis in vitro and to block the rejection of major histocompatibility complex (MHC)-mismatched mouse bone marrow cells. All compounds showed >99% binding to plasma proteins and demonstrated perforin inhibitory activity in vitro and in vivo. A lead compound, compound 1, that showed significant increases in allogeneic bone marrow preservation was evaluated for its plasma pharmacokinetics and in vivo efficacy at multiple dosing regimens to establish a pharmacokinetic/pharmacodynamic (PK/PD) relationship. The strongest PK/PD correlation was observed between perforin inhibition in vivo and time that total plasma concentrations remained above 900 µM, which correlates to unbound concentrations similar to 3× the unbound in vitro IC90 of compound 1. This PK/PD relationship will inform future dosing strategies of BZS perforin inhibitors to maintain concentrations above 3× the unbound IC90 for as long as possible to maximize efficacy and enhance progression toward clinical evaluation.

Inhibition of the Cytolytic Protein Perforin Prevents Rejection of Transplanted Bone Marrow Stem Cells in Vivo.

Perforin is a key effector protein in the vertebrate immune system and is secreted by cytotoxic T lymphocytes and natural killer cells to help eliminate virus-infected and transformed target cells. The ability to modulate perforin activity in vivo could be extremely useful, especially in the context of bone marrow stem cell transplantation where early rejection of immunologically mismatched grafts is driven by the recipient's natural killer cells, which overwhelmingly use perforin to kill their targets. Bone marrow stem cell transplantation is a potentially curative treatment for both malignant and nonmalignant disorders, but when the body recognizes the graft as foreign, it is rejected by this process, often with fatal consequences. Here we report optimization of a previously identified series of benzenesulfonamide-based perforin inhibitors for their physicochemical and pharmacokinetic properties, resulting in the identification of 16, the first reported small molecule able to prevent rejection of transplanted bone marrow stem cells in vivo by blocking perforin function.

Benzenesulphonamide inhibitors of the cytolytic protein perforin.

The pore-forming protein perforin is a key component of mammalian cell-mediated immunity and essential to the pathway that allows elimination of virus-infected and transformed cells. Perforin activity has also been implicated in certain auto-immune conditions and therapy-induced conditions such as allograft rejection and graft versus host disease. An inhibitor of perforin activity could be used as a highly specific immunosuppressive treatment for these conditions, with reduced side-effects compared to currently accepted therapies. Previously identified first-in-class inhibitors based on a 2-thioxoimidazolidin-4-one core show suboptimal physicochemical properties and toxicity toward the natural killer (NK) cells that secrete perforin in vivo. The current benzenesulphonamide-based series delivers a non-toxic bioisosteric replacement possessing improved solubility.

Tyrosine Kinase Inhibitors. 20. Optimization of Substituted Quinazoline and Pyrido[3,4-d]pyrimidine Derivatives as Orally Active, Irreversible Inhibitors of the Epidermal Growth Factor Receptor Family.

Structure-activity relationships for inhibition of erbB1, erbB2, and erbB4 were determined for a series of quinazoline- and pyrido[3,4-d]pyrimidine-based analogues of the irreversible pan-erbB inhibitor, canertinib. Cyclic amine bearing crotonamides were determined to provide rapid inhibition of cellular erbB1 autophosphorylation and good metabolic stability in liver microsome and hepatocyte assays. The influence of 4-anilino substitution on pan-erbB inhibitory potency was investigated. Several anilines were identified as providing potent, reversible pan-erbB inhibition. Optimum 4- and 6-substituents with known 7-substituents provided preferred irreversible inhibitors for pharmacodynamic testing in vivo. Quinazoline 54 and pyrido[3,4-d]pyrimidine 71 were identified as clearly superior to canertinib. Both compounds possess a piperidinyl crotonamide Michael acceptor and a 3-chloro-4-fluoroaniline, indicating these as optimized 6- and 4-substituents, respectively. Pharmacokinetic comparison of compounds 54 and 71 across three species selected compound 54 as the preferred candidate. Compound 54 (PF-00299804) has been assigned the nomenclature of dacomitinib and is currently under clinical evaluation.

A Selective and Slowly Reversible Inhibitor of l-Type Amino Acid Transporter 1 (LAT1) Potentiates Antiproliferative Drug Efficacy in Cancer Cells.

The l-type amino acid transporter 1 (LAT1) is a transmembrane protein carrying bulky and neutral amino acids into cells. LAT1 is overexpressed in several types of tumors, and its inhibition can result in reduced cancer cell growth. However, known LAT1 inhibitors lack selectivity over other transporters. In the present study, we designed and synthesized a novel selective LAT1 inhibitor (1), which inhibited the uptake of LAT1 substrate, l-leucin as well as cell growth. It also significantly potentiated the efficacy of bestatin and cisplatin even at low concentrations (25 µM). Inhibition was slowly reversible, as the inhibitor was able to be detached from the cell surface and blood-brain barrier. Moreover, the inhibitor was metabolically stable and selective toward LAT1. Since the inhibitor was readily accumulated into the prostate after intraperitoneal injection to the healthy mice, this compound may be a promising agent or adjuvant especially for the treatment of prostate cancer.

Systemic and Brain Pharmacokinetics of Perforin Inhibitor Prodrugs.

We have recently reported that by converting a perforin inhibitor into an l-type amino acid transporter 1 (LAT1)-utilizing prodrug its cellular uptake can be greatly increased. The aim of the present study was to determine the in vivo and brain pharmacokinetics of two perforin inhibitors and their LAT1-utilizing prodrugs 1 and 2. In addition, the brain uptake mechanism and entry into primary mouse cortical neurons and astrocytes were evaluated. After 23 µmol/kg i.p. bolus injection, the prodrugs' unbound area under the concentration curve in brain was 0.3 nmol/g × min, whereas the parent drugs could not reach the brain. The unbound brain concentrations of the prodrugs after 100 µM in situ mouse brain perfusion were 521.4 ± 46.9 and 126.9 ± 19.9 pmol/g for prodrugs 1 and 2, respectively. The combination of competing transporter substrates for LAT1, l-tryptophan, and for organic anion transporting polypeptides, probenecid, decreased the brain concentrations to 352.4 ± 44.5 and 70.9 ± 7.0 pmol/g, respectively. In addition, in vitro uptake studies showed that at 100 µM prodrug 1 had 3.4-fold and 4.5-fold higher uptake rate into neurons and astrocytes, respectively, compared to its parent drug. Thus, the prodrugs enhance significantly the therapeutic potential of the parent drugs for the treatment of disorders of central nervous system in which neuroinflammation is involved.

L-Type amino acid transporter 1 (lat1)-mediated targeted delivery of perforin inhibitors.

Perforin is a cytolytic pore-forming glycoprotein secreted by cytotoxic effector cells. It is a key component of the immune response against virus-infected and transformed cells and has been implicated in a number of human diseases. Perforin activity can be inhibited by small-molecular-weight compounds, although less is known about their delivery to the site of action. Therefore, in the present study, it was explored if perforin inhibitors could be efficiently and site-selectively delivered firstly into the cytotoxic effector cells and secondly into lytic granules, in which perforin is stored. This was accomplished by designing and synthesizing four prodrugs of perforin inhibitors that could utilize l-type amino acid transporter (LAT1), since activated immune cells are known to over-express LAT1. The results demonstrate that cellular uptake of perforin inhibitors can be increased by LAT1-utilizing prodrugs (into human breast adenocarcinoma cells (MCF-7)). Furthermore, these prodrugs were also able to deliver perforin inhibitors into the cell organelles having lower pH (rat liver lysosomes). Therefore, by using these prodrugs, intracellular mechanisms of perforin inhibitory activity can be studied more thoroughly in future. Moreover, this prodrug approach can be applied for other drugs that would benefit from targeted delivery into cells expressing LAT1, such as cancer.

Dihydrofuro[3,4-c]pyridinones as inhibitors of the cytolytic effects of the pore-forming glycoprotein perforin.

Dihydrofuro[3,4-c]pyridinones are the first class of small molecules reported to inhibit the cytolytic effects of the lymphocyte toxin perforin. A lead structure was identified from a high throughput screen, and a series of analogues were designed and prepared to explore structure-activity relationships around the core bicyclic thioxofuropyridinone and pendant furan ring. This resulted in the identification of a submicromolar inhibitor of the perforin-induced lysis of Jurkat T-lymphoma cells.

New small-molecule inhibitors of mitogen-activated protein kinase kinase.

BACKGROUND: Overexpression of the Ras/Raf/MEK/ERK (extracellular-signal-regulated kinase) pathway is associated with the formation, progression and survival of tumours and has also been implicated in a diverse range of therapeutic areas such as arthritis, organ transplant rejection, asthma and developmental disorders. One approach to down regulation of this pathway is through the inhibition of mitogen-activated protein kinase kinase 1/2 (MEK1/2). OBJECTIVE: The importance of the mitogen-activated protein kinase (MAPK) pathway, MEK1/2 as a therapeutic target and early MEK1/2 inhibitors is discussed, followed by an overview of recent patent activity in the area. METHODS: The patent literature was searched for inhibitors of MEK1/2 published within the last three years; these results are described. Other relevant publications that provide further insight into the discovery and development of these compounds are also discussed. CONCLUSION: The determination of a crystal structure with inhibitor bound has allowed the design of exquisitely selective and potent inhibitors of MEK1/2. Several allosteric inhibitors have advanced to clinical trial and shown some efficacy in cancer as single agents, but the future application of MEK1/2 inhibitors is likely to be either in combination with other therapies or in disorders which are genetically defined as being dependent on the MAPK pathway.

4-anilino-5-carboxamido-2-pyridone derivatives as noncompetitive inhibitors of mitogen-activated protein kinase kinase.

A new series of MEK1 inhibitors, the 4-anilino-5-carboxamido-2-pyridones, were designed and synthesized using a combination of medicinal chemistry, computational chemistry, and structural elucidation. The effect of variation in the carboxamide side chain, substitution on the pyridone nitrogen, and replacement of the 4'-iodide were all investigated. This study afforded several compounds which were either equipotent or more potent than the clinical candidate CI-1040 (1) in an isolated enzyme assay, as well as murine colon carcinoma (C26) cells, as measured by suppression of phosphorylated ERK substrate. Most notably, pyridone 27 was found to be more potent than 1 in vitro and produced a 100% response rate at a lower dose than 1, when tested for in vivo efficacy in animals bearing C26 tumors.

Structure-activity relationships for pyrido-, imidazo-, pyrazolo-, pyrazino-, and pyrrolophenazinecarboxamides as topoisomerase-targeted anticancer agents.

Heterocyclic phenazinecarboxamides were prepared by condensation of aminoheterocycles and 2-halo-3-nitrobenzoic acids, followed by reductive ring closure and amidation. They showed similar inhibition of paired cell lines that underexpressed topo II or overexpressed P-glycoprotein, indicating a non topo II mechanism of cytotoxicity and indifference to P-glycoprotein mediated multidrug resistance. Compounds with a fused five-membered heterocyclic ring were generally less potent than the pyrido[4,3-a]phenazines. A 4-methoxypyrido[4,3-a]phenazine (IC(50)s 2.5-26 nM) gave modest (ca. 5 day) growth delays in H69/P xenografts with oral dosing.

Synthesis and evaluation of unsymmetrical bis(arylcarboxamides) designed as topoisomerase-targeted anticancer drugs.

Symmetrical dimers of lipophilic intercalating chromophores linked by cation-containing chains have recently been shown to have broad-spectrum in vivo anticancer activity. We report the preparation and evaluation of a series of both symmetric and unsymmetric dimers of a variety of intercalating chromophores of varied DNA binding strength, including naphthalimides, acridines, phenazines, oxanthrenes and 2-phenylquinolines. The unsymmetrical dimers were prepared by sequential coupling of the chromophores to linkers with selectively protected primary terminal amines to ensure high yields and unequivocal product. Protection of the internal (secondary) amines as BOC derivatives was used to ensure complete structural specificity, and was also an aid to the purification of these very polar compounds. The growth inhibitory abilities (as IC(50) values) of the compounds in a range of cell lines showed that the nature of the linker chain was important, and independent of the nature of the chromophore, with compounds containing the dicationic linker [-(CH2)2NH(CH2)2NH(CH2)2-] being on average 30-fold more potent than the corresponding compounds containing the monocationic linker [-(CH2)3NMe(CH2)3-]. However, the chromophores also play a role in determining biological activity, with the cytotoxicities of symmetric and unsymmetric dicationic dimers correlating with the overall DNA binding abilities of the chromophores.