{"id":146,"date":"2019-04-11T16:40:24","date_gmt":"2019-04-11T20:40:24","guid":{"rendered":"https:\/\/sites.bu.edu\/gareth-morgan\/?p=146"},"modified":"2019-04-11T16:40:24","modified_gmt":"2019-04-11T20:40:24","slug":"new-paper-small-molecules-that-stabilize-light-chains","status":"publish","type":"post","link":"https:\/\/sites.bu.edu\/gareth-morgan\/2019\/04\/11\/new-paper-small-molecules-that-stabilize-light-chains\/","title":{"rendered":"New paper: Small molecules that stabilize light chains"},"content":{"rendered":"

A major goal of the lab is to develop new therapies for amyloidosis. Today, our paper<\/a> describing a big step towards a drug for AL amyloidosis was published. We have identified small molecules that bind to antibody light chains and prevent them from unfolding, which could potentially reduce amyloid formation in patients. We\u2019re a long way from a drug yet, but this paper shows where and how a drug might bind. One of the molecules is a simple fluorescent dye that could be a useful tool for further studies. Along the way, we figured out a new way to screen large libraries of molecules to find stabilizers. This was a big team effort, mostly carried out at Scripps Research<\/a> during my time as a postdoc there, and I especially want to thank co-authors Jeff Kelly and Nick Yan, who put a huge amount of work into making this happen.<\/p>\n

(I do have a conflict of interest here: we have applied for a patent for this work and there\u2019s a chance that I might make some money from sales of an eventual drug.)<\/p>\n

AL amyloidosis<\/a> can be treated by killing the clonal plasma cells that secrete the antibody light chains that aggregate to cause disease. However, the treatments are very harsh and don\u2019t work for everyone. If we can prevent the misfolding of light chains, independently of the cells that produce them, we may be able to stabilize patients and improve their quality of life. Ideally, this strategy would allow sick patients to tolerate chemotherapy.<\/p>\n

Amyloidosis can be caused by destabilization of the precursor protein, which encourages the structural changes that lead to aggregation. In transthyretin amyloidosis, this is caused by hereditary mutations or ageing. Stabilizing transthyretin using small molecules<\/a> (tafamidis, diflunisal or AG10) prevents misfolding and can prevent disease progression. In AL amyloidosis, each patient has a different light chain sequence but there\u2019s evidence that less stable light chains are more likely to cause disease. Our work on full-length light chains<\/a> suggested that stabilizing these proteins, rather than the isolated variable domains that most other groups had focused on, could be helpful.<\/p>\n

The Kelly lab at Scripps developed tafamidis as a stabilizer of transthyretin, so we have some experience with this kind of thing. Transthyretin is a hormone transport protein with a deep binding pocket for its natural ligand, thyroxin. Unfortunately, light chains don\u2019t have known ligands or any obvious binding pockets, so we needed to start from scratch.<\/p>\n

The standard way to find drugs for an unknown target is to use a high-throughput screen. Here, a measurement is repeated thousands of times in parallel, with a different small molecule in each reaction. The key to successful screening is to start with a large library of small molecules, which means robotic liquid handling and 1,536-well plates. Screening for compounds that enhance stability is difficult, because the methods used in most laboratories can\u2019t be used on microwell plates.<\/p>\n

We developed a method to get around this based on proteolysis, using fluorescence polarization as a readout. Josh Blunden, an undergraduate student who worked in the lab in the summer of 2016, did a lot of the legwork needed to get this started. Then Steve Brown at Scripps helped turn it into a working assay that we could use to screen the compound library at Scripps Florida. The assay works well and it could be useful for other targets. Somewhat surprisingly, no-one seems to have done this before. We\u2019re intending to publish a methods paper to help other groups use it.<\/p>\n

\"A<\/a>
Our screening method uses fluorescence polarization to detect proteolysis of a fluorescein-labeled protein.<\/figcaption><\/figure>\n

This is where my part in the story could have ended. High throughput screening is expensive (we used a thousand or so plates, at five dollars each, for example). Getting a grant to fund the screen might have taken a year or more. Fortunately, Jeff was sufficiently enthusiastic about the prospects of developing a drug that he was able to scrape the money together and say \u201cMake it so!\u201d It\u2019s a huge commitment that wouldn\u2019t have been possible in most labs and I\u2019m really grateful that we were able to go for it.<\/p>\n

Tim Spicer and Virneliz Fernandez-Vega at Scripps Florida<\/a> screened 650,000 compounds and identified 2,777 hits. Screens tend to throw up a lot of false positives, so we used secondary screens to narrow the list down to 128 molecules that they shipped to us. We picked out the best 16 molecules, which could be divided into four chemical classes. Finally, we chose one molecule for detailed characterization.<\/p>\n

What\u2019s so special about the final molecule? Part of the answer is that we had a bottle of it in the cupboard \u2013 it\u2019s a commercially-available<\/a> fluorescent dye called coumarin 1. All the best molecules had similar activity, so we picked this one because it was convenient and readily available to other researchers. We do have some evidence that the other molecules bind in the same way, so we were able to use coumarin 1 as a tool to ask about the mechanisms and consequences of stabilizer binding. We verified that coumarin 1 and the other small molecules stabilize multiple light chains against different types of perturbations, so we\u2019re confident that it works in the way we were aiming for.<\/p>\n

At the end of 2017, Nick Yan joined the lab as a graduate student. He quickly took on the project of characterizing the small molecule hits from the screen. With the help of David Mortenson in the Kelly lab, and Ian Wilson\u2019s structural biology group at Scripps, Nick solved two crystal structures: a light chain protein alone<\/a>, and the same light chain in the presence of coumarin 1<\/a>. The structures show where the small molecule binds \u2013 between the two variable domains of the light chain dimer. We verified that light chains bind to coumarin 1 in the same way in solution using NMR, in collaboration with Enrico Rennella and Lewis Kay in Toronto, who have collaborated with us to understand the dynamics of the light chain dimer<\/a>.<\/p>\n

\"Structure<\/a>
Coumarin 1 bound to a full-length antibody light chain dimer. Constant domains are grey and variable domains are blue.<\/figcaption><\/figure>\n

What\u2019s really encouraging and unexpected about the binding site is that it\u2019s built from residues that are very similar between different light chains. Each AL amyloidosis patient has a unique protein sequence, so a potential drug needs to bind at a site that\u2019s conserved between different light chains. That\u2019s what we found \u2013 the binding site is made up of residues that form the structural core of the variable domains. The same residues are present in more than 90% of all light chains. Coumarin 1 binds into a pocket that\u2019s not present in the unliganded native structure, a so-called \u201ccryptic\u201d binding site. Importantly, the binding site is not present in normal antibody heavy chain-light chain dimers, which means that the molecules should be specific for free light chains.<\/p>\n

Coumarin 1 is not an ideal drug molecule. It doesn\u2019t bind tightly enough to the light chain, and it will probably stick to a lot of other molecules in the body. We\u2019re more excited about the binding site that we discovered. We will make molecules that bind much more tightly and specifically to light chains, based on the structure of the complex.<\/p>\n

On the other hand, coumarin 1 is a fluorescent dye, and its fluorescence gets much brighter when it\u2019s bound to light chains than when it\u2019s free in solution. This means that we can use it as a probe for folded light chain dimers. This is not something that\u2019s written on the bottle and it wouldn\u2019t necessarily have been an obvious thing to try. But this \u201cfluorogenicity\u201d has been a big part of the lab\u2019s research<\/a> and it\u2019s such a useful property that we routinely check for it in new compounds. In the paper, we use this property to ask how tightly the small molecule binds to different light chains, and whether other molecules bind in the same site. We hope that this will be a useful tool for other amyloidosis researchers.<\/p>\n

I\u2019ve spent three years working on this project and it\u2019s great to see it published at last. It\u2019s really the beginning of a process that we hope will lead to a drug for what is often an untreatable disease. There are a lot of challenges before we can think about clinical trials, but I\u2019m confident that we will be able to make progress.<\/p>\n","protected":false},"excerpt":{"rendered":"

A major goal of the lab is to develop new therapies for amyloidosis. Today, our paper describing a big step towards a drug for AL amyloidosis was published. We have identified small molecules that bind to antibody light chains and prevent them from unfolding, which could potentially reduce amyloid formation in patients. We\u2019re a long […]<\/p>\n","protected":false},"author":15684,"featured_media":0,"comment_status":"closed","ping_status":"open","sticky":false,"template":"","format":"standard","meta":[],"categories":[1],"tags":[9,19,12,10,11,16],"_links":{"self":[{"href":"https:\/\/sites.bu.edu\/gareth-morgan\/wp-json\/wp\/v2\/posts\/146"}],"collection":[{"href":"https:\/\/sites.bu.edu\/gareth-morgan\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/sites.bu.edu\/gareth-morgan\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/sites.bu.edu\/gareth-morgan\/wp-json\/wp\/v2\/users\/15684"}],"replies":[{"embeddable":true,"href":"https:\/\/sites.bu.edu\/gareth-morgan\/wp-json\/wp\/v2\/comments?post=146"}],"version-history":[{"count":5,"href":"https:\/\/sites.bu.edu\/gareth-morgan\/wp-json\/wp\/v2\/posts\/146\/revisions"}],"predecessor-version":[{"id":153,"href":"https:\/\/sites.bu.edu\/gareth-morgan\/wp-json\/wp\/v2\/posts\/146\/revisions\/153"}],"wp:attachment":[{"href":"https:\/\/sites.bu.edu\/gareth-morgan\/wp-json\/wp\/v2\/media?parent=146"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/sites.bu.edu\/gareth-morgan\/wp-json\/wp\/v2\/categories?post=146"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/sites.bu.edu\/gareth-morgan\/wp-json\/wp\/v2\/tags?post=146"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}