LAT1 transporters expressed in blood brain barrier cells
View Patent ↗LAT1 is consistently expressed at high levels in brain microvessel endothelial cells. Disclosed herein are assays for determining whether a test material/molecule is a substrate for, and/or is actively transported by, the LAT1 transporter, and therefore a candidate substrate for crossing the blood brain barrier. The assays are useful in screening for therapeutic, cytotoxic or imaging compounds used in the treatment or diagnosis of neurological diseases.
1. A method of screening an agent, conjugate or conjugate moiety for capacity to be transported through the blood brain barrier, comprising:
(a) providing a cell expressing a LAT1 transporter, wherein:
a nucleic acid molecule encoding the LAT1 transporter has been transfected or injected into the cell; and
the LAT1 transporter is the protein encoded by SEQ ID NO: 1 or has at least 90% sequence identity to the protein encoded by SEQ ID NO:1, and the LAT1 transporter can transport phenylalanine, the LAT1 transporter being situated in the plasma membrane of the cell;
(b) contacting the cell with an agent, conjugate or conjugate moiety in vitro; and
(c) determining whether the agent, conjugate or conjugate moiety passes through the plasma membrane via the LAT1 transporter, passage through the plasma membrane via the LAT1 transporter indicating the agent, conjugate or conjugate moiety has capacity to be transported through the blood brain barrier,
wherein:
if step (b) comprises contacting the cell with the agent, the agent is a neuropharmaceutical agent or an imaging component;
if step (b) comprises contacting the cell with the conjugate, the conjugate comprises an agent that is a neuropharmaceutical agent or an imaging component; or
if step (b) comprises contacting the cells with the conjugate moiety, the method further comprises linking the conjugate moiety to an agent that is a neuropharmaceutical agent or an imaging component.
2. The method of claim 1 , wherein the cell also expresses a 4F2HC protein.
3. The method of claim 1 , wherein the cell is an oocyte.
4. The method of claim 1 , wherein the determining is performed by a competition assay.
5. The method of claim 1 , wherein the determining is performed by an exchange assay.
6. The method of claim 1 , wherein the determining is performed by a direct uptake assay.
7. The method of claim 1 , wherein the cell is a human embryonic kidney (HEK) cell.
8. The method of claim 1 , wherein the cell is transformed with an SV40 large T antigen that can be expressed in a temperature sensitive fashion.
9. The method of claim 1 , wherein the determining step determines that the agent, conjugate or conjugate moiety passes through the plasma membrane via the LAT1 transporter; and the method further comprises:
(d) modifying the agent, conjugate or conjugate moiety; and
(e) determining if the modified agent, conjugate or conjugate moiety is transported with a higher V max by the LAT1 transporter than the agent, conjugate or conjugate moiety.
10. The method of claim 1 , wherein the neuropharmaceutical agent is a cytotoxic neuropharmaceutical agent selected from the group consisting of platinum, nitrosourea, nitroimidazole, and nitrogen mustard.
11. The method of claim 1 , wherein the agent, conjugate or conjugate moiety comprises an amino acid.
12. The method of claim 11 , wherein the amino acid is selected from the group consisting of tryptophan, leucine, methionine, phenylalanine, bicyclohexane amino acid, L-dopa, gabapentin, and baclofen.
13. The method of claim 1 , further comprising:
(d) providing a cell expressing at least one efflux transporter;
(e) contacting the cell with the agent, conjugate or conjugate moiety in vitro;
(f) determining that the agent, conjugate or conjugate moiety is transported by to the protein encoded by SEQ ID NO:1 at least one efflux transporter selected from the group consisting of the P-glycoprotein transporter, the multidrug resistance protein transporter, and the breast cancer resistance protein transporter; and
determining the efflux transporter activity of the agent, conjugate or conjugate moiety.
14. The method of claim 13 , further comprising:
(g) determining the LAT1 transporter activity of the agent, conjugate or conjugate moiety;
(h) modifying the agent, conjugate or conjugate moiety;
(i) establishing that the modified agent, conjugate or conjugate moiety retains LAT1 activity; and
determining the LAT1 transporter activity of the modified agent, conjugate or conjugate moiety;
(k) determining the efflux transporter activity of the modified agent, conjugate or conjugate moiety; and
(l) comparing the ratio of LAT 1 activity to the ratio of efflux transporter activity for the agent, conjugate or conjugate moiety and the ratio of LAT1 transporter activity to the efflux transporter activity of the modified agent, conjugate or conjugate moiety wherein an increased ratio of LAT1 activity to efflux transporter activity demonstrates that the modification improves the capacity of the agent, conjugate or conjugate moiety to be transported through the blood brain barrier.
15. The method of claim 13 or 14 , wherein the efflux transporter activity is determined by conducting an assay selected from the group consisting of:
(a) an efflux transporter ATPase activity assay;
(b) an efflux transporter competition assay; and
(c) a direct efflux transport assay across a polarized monolayer of cells.
16. The method of claim 13 or 14 , wherein the efflux transporter activity is the V max for the efflux transporter.
17. The method of claim 14 , wherein the LAT1 transporter activity is the V max for the LAT1 transporter.
18. A method of using the LAT1 transporter to identify an agent or conjugate having the capacity to be transported through the blood brain barrier, comprising:
(a) providing a cell expressing the LAT1 transporter, wherein:
a nucleic acid molecule encoding the LAT1 transporter has been tranfected or injected into the cell; and
the LAT1 transporter in the protein encoded by SEQ ID NO:1 or has at least 90% sequence identity thereto, and the LAT1 transporter can transport phenylalanine, the LAT1 transporter being situated in the plasma membrane of the cell;
(b) contracting the cell with an agent, conjugate or conjugate moiety in vitro;
(c) determining that the agent or conjugate is a substrate for the LAT1 transporter; and
(d) determining that the agent or conjugate is actively transported by the LAT1 transporter; transport by the LAT1 transporter indicating that the agent or conjugate has has capacity to be transported through the blood brain barrier.