Small Molecule Kinetic Stabilizers Reduce Amyloidogenicity of Free Light Chain Proteins of Diverse Sequences in λ Light Chain Amyloidosis

In light chain amyloidosis (AL), clonal plasma cells produce free immunoglobulin light chain (FLC) proteins that are unstable and prone to misfolding, fragmentations, and aggregation, primarily affecting the heart and kidney. This instability in FLC is not just a characteristic of the disease but also the root cause of toxicity and the main contributor to amyloidosis-related morbidity and mortality in AL. The involved λFLC accounts for ~75-80% of all AL cases. λFLC usually forms a disulfide-linked homodimer in circulation, although co-existing monomers are also commonly detected.

All current therapies aim to minimize FLC-producing clonal plasma cells, while a couple of development candidates target amyloid fibrils. However, none of these directly address the soluble toxic protein conformations associated with the amyloidosis aspect of the disease, which is believed to be a strong contributor to the pathology, based on existing data on AL and other amyloidosis. To tackle the toxicity elicited by unstable FLC, we adopted the kinetic stabilizer approach previously used successfully to treat other amyloidoses, such as tafamidis for transthyretin amyloidosis.

A unique challenge in developing an FLC kinetic stabilizer lies in the fact that FLC proteins exhibit significant sequence diversity, primarily due to somatic recombination in the variable domain (V-region). Each AL patient has a unique clonal FLC sequence. Small molecule kinetic stabilizers were previously reported to bind through an induced-fit pocket formed by conserved amino acids at the λFLC variable domain interface and protect the amyloidogenic WIL protein from limited proteolytic degradation. However, we found these early compounds did not exhibit sufficient selectivity for λFLC in plasma.

To address these limitations, we explored the structure-activity relationships by incorporating X-ray crystallographic data and targeted modifications to optimize for λFLC plasma selectivity. The structural insights provided by the X-ray structures enabled the rational design of pyridone-based FLC kinetic stabilizers and their analogs. This iterative process significantly improved compound potency and specificity, enabling FLC stabilization in plasma.

At the same time, we optimized λFLC stabilizers sequence coverage and compound binding properties. An analysis of amyloidogenic λFLC sequences from the AL-Base (https://wwwapp.bumc.bu.edu/BEDAC_ALBase/) indicates that AL sequences are concentrated within four subfamilies of the V-region, v1, v2, v3, and v6, with the other subfamilies uncommon. Recombinant amyloidogenic λFLC protein sequences, majority from the four subfamilies, were selected from the AL-Base and produced to represent the breadth of λAL FLC sequences. Differential Scanning Fluorimetry (DSF) and limited proteolysis were used to characterize the stability of the recombinant λFLC proteins with or without λFLC kinetic stabilizers. The small molecule stabilizers were able to stabilize most of a cohort of 12 recombinant amyloidogenic λFLC proteins thermodynamically and kinetically. For example, PTGP-150 was able to dose-dependently stabilize all 12 amyloidogenic λFLC proteins with DTm in DSF ranging from 0.3°C to 6.3°C, with 11 of the proteins achieving DTm>2°C. Similarly, 11 of the 12 λFLC proteins were protected against limited proteolytic degradation in a buffer environment.

The AmyLite™ assay was then used to measure the level of unstable FLC in ex vivo biofluids through the quantification of a novel biomarker dLCCD (dimeric light chain constant domain), of which the baseline levels in λAL patient plasmas strongly correlate to overall survival. We showed that adding PTGP-150 to ex vivo plasma could protect endogenous amyloidogenic λFLC in many patient plasma samples and potently reduce the protein's toxicity.

In summary, we show optimized small molecule FLC kinetic stabilizers can improve the kinetic and thermodynamic stability of amyloidogenic λFLC proteins of diverse sequences and protect them from limited proteolysis, potentially reducing the amyloidogenicity/toxicity of FLC in λAL patient plasmas. Small molecule kinetic stabilizer targeted therapy for AL represents a new mechanism of action explicitly aiming at reducing the toxicity of the amyloidogenic λFLC, offering promise for more effective treatment options for patients suffering from this debilitating condition.