Nature: Folate transporters provide clues to anticancer drugs or drive the success of cancer immunotherapy

Cyclic dinucleotides (CDN) are widely used and important messenger molecules
in nature.
Mammalian cells produce a specific CDN, 2'3'-cGAMP, which is catalyzed by the natural immune receptor cGAS after sensing abnormal DNA signals in the cytoplasm
.
2'3'-cGAMP binds to and activates downstream linker protein STING, thereby inducing a broad-spectrum immune response
.
In addition to 2'3'-cGAMP, a variety of CDNs secreted by pathogenic bacteria and widely concerned CDN-based anticancer drugs can activate STING and regulate immune response
.

One of the concerns in the field is how polar CDN molecules enter cells across membranes? Studies have shown that the solute carrier family protein SLC19A1 is a key CDN transporter
.
In addition to CDN, SLC19A1 is a major folate and folate-resistant transporter
.
Interestingly, some antifolate drugs are often used to treat some autoimmune diseases
through some "miraculous and unknown mechanism.
" SLC19A1 is speculated to play a role
in this process because it can transport these two types of substrates at the same time and inhibit each other the transport of these two types of substrates.

Given the important functions of SLC19A1 in CDN, folic acid and antifolic acid transmembrane transport, the study of its substrate recognition mechanism will help the mechanistic analysis of SLC19A1-related diseases and the development and optimization
of potential drugs.

Recently, Gao Pu's team from the Institute of Biophysics of the Chinese Academy of Sciences, Zhang Liguo's team and the Gao team of Beijing Institute of Technology published a research paper on Recognition of cyclic dinucleotides and folates by human SLC19A1 online in the journal Nature.

The study demystifies SLC19A1's identification of different substrate molecules, and insights into the structure of SLC19A1 proteins may lead to entirely new targeted anti-cancer therapies or promote the success of
cancer immunotherapy.

The research team first performed a large number of screens and obtained a series of murine monoclonal antibodies that identify SLC19A1 to stabilize protein samples
.
Using one of the rigidly binding antibodies, the researchers then resolved the substrate-free EMM structure
of SLC19A1 to the inward-open of the cell.
SLC19A1 is an MFS-type transporter composed of 12 transmembrane helixes that identifies key components and intramolecular interactions in the SLC19A1 inward-open conformation
.
The lumen and cytosolic inlet of SLC19A1 substrate carry a large amount of positive charge, which is consistent
with the anionic characteristics of the substrate transported by SLC19A1.
By mapping reported loss-of-function or disease-related mutations, the team found that most of these mutations affected substrate recognition, transport, or protein self-folding
.

Next, the research team analyzed the electron microscopy structure
of SLC19A1 with three CDN complexes from different sources in the ward-open state.
Surprisingly, all three CDN molecules bind to the positively charged lumen bottom of SLC19A1 by forming tight and delicate dimer units, a substrate recognition method that has not been reported
in other SLC or MFS family proteins.
The CDN dimer unit presents a conformation of bases in the middle and phosphate sugar rings at both ends, and this special assembly method relies on stacking and hydrogen bonding interaction between the two CDN molecules
.
Although the binding patterns of different CDNs are roughly similar, there are obvious conformational differences in their respective dimer units, which reflects the broad inclusiveness
of SLC19A1 for different CDNs.

In order to explore the mechanism by which SLC19A1 recognizes folic acid and antifolic acid, the research team successfully resolved the high-resolution electron microscopy structure
of SLC19A1 with 5-MTHF (reduced folic acid mainly present in diet and blood) and PMX (a new class of antifolate drugs) complexes using a new set of antioxidants.
However, unlike CDNs, both 5-MTHF and PMX are bound as monomers in the upper middle part of the polar cavity of SLC19A1
.
SLC19A1 was previously known to transport reduced folate much more efficiently than ordinary folate, and the researchers found that SLC19A1 has an additional interaction
with the 5-position methyl and 8 hydrogen atoms of 5-MTHF.

Although CDN and folate/antifolate bind to SLC19A1 at completely different sites, both substrates are transported through the same internal channel, so there is clearly competitive inhibition
between the two.
The researchers systematically analyzed the two substrate binding pockets and found that some mutations affected the transport of both types of substrates at the same time, while some mutations only had a significant effect on the transport of one type of substrate, suggesting that it is possible to design small molecule drugs
with substrate inhibition selectivity.

SLC19A1 transport CDN(a) and folate/folic acid (b) conceptual map and model map

In summary, this study reports the molecular basis of human SLC19A1 for recognizing different substrates such as CDN, folic acid and folic acid resistance, reveals its unique and diverse substrate recognition mechanism, and identifies a novel substrate recognition mode
of SLC and MFS families.
This study provides new ideas for the development of a new generation of CDN drugs and antifolic acid drugs, and the antibody screening platform established in the study and the multiple monoclonal antibodies obtained also provide a basis
for the development of antibody drugs.