Umami is one of the 5 basic taste qualities. The umami taste of L-glutamate can be drastically enhanced by 5 ribonucleotides and the synergy is a hallmark of this taste quality. The umami taste receptor is a heteromeric complex of 2 class C G-protein-coupled receptors, T1R1 and T1R3. Here we elucidate the molecular mechanism of the synergy using chimeric T1R receptors, site-directed mutagenesis, and molecular modeling. We propose a cooperative ligand-binding model involving the Venus flytrap domain of T1R1, where L-glutamate binds close to the hinge region, and 5 ribonucleotides bind to an adjacent site close to the opening of the flytrap to further stabilize the closed conformation. This unique mechanism may apply to other class C G-protein-coupled receptors.glutamate ͉ G protein-coupled receptors ͉ IMP ͉ T1R H umans can detect at least 5 basic taste qualities, including sweet, umami, bitter, salty, and sour. Umami, the savory taste of L-glutamate, was first discovered in 1908 by K. Ikeda, but only recently accepted as a basic taste quality by the general public. The most unique characteristic of umami taste is synergism. Purinic ribonucleotides, such as IMP and GMP, can strongly potentiate the umami taste intensity (1). In human taste tests, 200 M of IMP, which does not elicit any umami taste by itself, can increase one's umami taste sensitivity to glutamate by 15-fold (2).Among the 5 basic taste qualities, sweet, umami, and bitter taste are mediated by G protein-coupled receptors (GPCRs) (3). Receptors for umami taste and sweet taste are closely related to each other. The 3 subunits of the T1R family form 2 heteromeric receptors: umami (T1R1/T1R3) (2, 4) and sweet (T1R2/T1R3) (2, 5). T1R receptors belong to the class C GPCRs, along with metabotropic glutamate receptors (mGluRs), ␥-aminobutyric acid receptor B (GABA B R), calcium sensing receptors (CaSR), and so forth. The defining motif in these receptors is an outer membrane N-terminal Venus flytrap (VFT) domain that consists of 2 globular subdomains, the N-terminal upper lobe and the lower lobe, that are connected by a 3-stranded flexible hinge. The VFT domain of C-GPCRs contains the ligand-binding site (6), as demonstrated by studies on mGluRs, GABA B R, and the sweet taste receptor (7). The crystal structures of mGluR VFT domains revealed that the bi-lobed architecture can form an open or closed conformation (8, 9). Glutamate binding stabilizes both the active dimer and the closed conformation. This scheme in the initial receptor activation has been applied generally to other C-GPCRs.Studies on sweet taste-receptor functional domains revealed multiple binding sites for its structurally diverse ligands. Using human-rat chimeric receptors, we demonstrated the T1R2 VFT domain of the human sweet receptor interacts with 2 structurally related synthetic sweeteners aspartame and neotame, while the transmembrane domain (TMD) of human T1R3 interacts with another sweetener cyclamate and a human sweet-taste inhibitor lactisole (7). Works from several other laborator...

Positive allosteric modulators of the human sweet taste receptor have been developed as a new way of reducing dietary sugar intake. Besides their potential health benefit, the sweet taste enhancers are also valuable tool molecules to study the general mechanism of positive allosteric modulations of T1R taste receptors. Using chimeric receptors, mutagenesis, and molecular modeling, we reveal how these sweet enhancers work at the molecular level. Our data argue that the sweet enhancers follow a similar mechanism as the natural umami taste enhancer molecules. Whereas the sweeteners bind to the hinge region and induce the closure of the Venus flytrap domain of T1R2, the enhancers bind close to the opening and further stabilize the closed and active conformation of the receptor.positive allosteric modulators | sweet taste receptor | T1R H umans can detect at least five basic taste qualities, including sweet, umami, bitter, salty, and sour. The sweet and umami taste are mediated by closely related G protein-coupled receptors (GPCRs). The three members of the T1R family form two heteromeric taste receptors: umami (T1R1/T1R3) (1, 2) and sweet (T1R2/T1R3) (1, 3). T1R receptors belong to the class C GPCRs, along with metabotropic glutamate receptors (mGluRs), γ-aminobutyric acid receptor B (GABA B R), calcium sensing receptors (CaSR), and others. The defining motif in these receptors is an extracellular Venus flytrap (VFT) domain (4), which consists of two globular subdomains connected by a threestranded flexible hinge. The VFT domain contains the orthosteric ligand binding site. The crystal structures of mGluR VFT domains (5, 6) revealed that the bilobed architecture can form an "open" or "closed" conformation. Glutamate binding stabilizes both the "closed" and the "active" dimer conformation. This scheme in the initial receptor activation has been applied generally to other class C GPCRs.Over the years, researchers have been developing noncaloric sweeteners to reduce dietary sugar intake. Unfortunately, all existing noncaloric sweeteners are characterized by their off taste (7,8) and fail to mimic the real sugar taste. Since the identification of the sweet taste receptor, a new approach became available, which is to develop positive allosteric modulators (PAMs) of the receptor. These molecules work as sweet taste "enhancers," which possess no taste of their own but potentiate the sweet taste of sugars. Examples of taste enhancers can be found in umami taste, which is known for its unique characteristic of synergism (9). Purinic ribonucleotides such as inosine-5′-monophosphate (IMP) and guanosine-5′-monophosphate (GMP) can strongly potentiate the umami taste intensity of glutamate and are rare examples of naturally occurring GPCR PAMs. In taste tests, 200 μM IMP, which does not elicit any umami taste by itself, can increase human umami taste sensitivity to glutamate by 15-fold (1). We recently illustrated the molecular mechanism of IMP/GMP (10). Our data indicate that glutamate binds close to the hinge region of the VFT ...

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Chronic myelogenous leukemia (CML) is a hematological stem cell disorder caused by increased and unregulated growth of myeloid cells in the bone marrow, and the accumulation of excessive white blood cells. Abelson tyrosine kinase (ABL) is a non-receptor tyrosine kinase involved in cell growth and proliferation and is usually under tight control. However, 95% of CML patients have the ABL gene from chromosome 9 fused with the breakpoint cluster (BCR) gene from chromosome 22, resulting in a short chromosome known as the Philadelphia chromosome. This Philadelphia chromosome is responsible for the production of BCR-ABL, a constitutively active tyrosine kinase that causes uncontrolled cellular proliferation. An ABL inhibitor, imatinib, was approved by the FDA for the treatment of CML, and is currently used as first line therapy. However, a high percentage of clinical relapse has been observed due to long term treatment with imatinib. A majority of these relapsed patients have several point mutations at and around the ATP binding pocket of the ABL kinase domain in BCR-ABL. In order to address the resistance of mutated BCR-ABL to imatinib, 2(nd) generation inhibitors such as dasatinib, and nilotinib were developed. These compounds were approved for the treatment of CML patients who are resistant to imatinib. All of the BCR-ABL mutants are inhibited by the 2(nd) generation inhibitors with the exception of the T315I mutant. Several 3(rd) generation inhibitors such as AP24534, VX-680 (MK-0457), PHA-739358, PPY-A, XL-228, SGX-70393, FTY720 and TG101113 are being developed to target the T315I mutation. The early results from these compounds are encouraging and it is anticipated that physicians will have additional drugs at their disposal for the treatment of patients with the mutated BCR-ABL-T315I. The success of these inhibitors has greater implication not only in CML, but also in other diseases driven by kinases where the mutated gatekeeper residue plays a major role.

Age-related macular degeneration (AMD) is one of the leading causes of loss of vision in the industrialized world. Attenuating the VEGF signal in the eye to treat AMD has been validated clinically. A large body of evidence suggests that inhibitors targeting the VEGFr pathway may be effective for the treatment of AMD. Recent studies using Src/YES knockout mice suggest that along with VEGF, Src and YES play a crucial role in vascular leak and might be useful in treating edema associated with AMD. Therefore, we have developed several potent benzotriazine inhibitors designed to target VEGFr2, Src, and YES. One of the most potent compounds is 4-chloro-3-{5-methyl-3-[4-(2-pyrrolidin-1-yl-ethoxy)phenylamino]benzo[1,2,4]triazin-7-yl}phenol ( 5), a dual inhibitor of both VEGFr2 and the Src family (Src and YES) kinases. Several ester analogues of 5 were prepared as prodrugs to improve the concentration of 5 at the back of the eye after topical administration. The thermal stability of these esters was studied, and it was found that benzoyl and substituted benzoyl esters of 5 showed good thermal stability. The hydrolysis rates of these prodrugs were studied to analyze their ability to undergo conversion to 5 in vivo so that appropriate concentrations of 5 are available in the back-of-the-eye tissues. From these studies, we identified 4-chloro-3-(5-methyl-3-{[4-(2-pyrrolidin-1-ylethoxy)phenyl]amino}-1,2,4-benzotriazin-7-yl)phenyl benzoate ( 12), a topically administered prodrug delivered as an eye drop that is readily converted to the active compound 5 in the eye. This topically delivered compound exhibited excellent ocular pharmacokinetics and poor systemic circulation and showed good efficacy in the laser induced choroidal neovascularization model. On the basis of its superior profile, compound 12 was advanced. It is currently in a clinical trial as a first in class, VEGFr2 targeting, topically applied compound for the treatment of AMD.