Periodic Reporting for period 1 - MEGAKINE (Megakines - novel tools for structural insight into receptor:chemokine complexes)
Reporting period: 2021-09-01 to 2023-08-31
Chemokines are small, secreted cytokines that activate membrane receptors belonging to the G protein-coupled receptor (GPCR) superfamily. The chemokine-receptor interactions control crucial physiological processes, but are also implicated in many pathologies, including atherosclerosis, inflammatory diseases, HIV infection or cancer, and hold therefore a great potential for therapeutic intervention.
Yet, although GPCRs are the target of about one third of currently marketed drugs, only three of them act on chemokine receptors, essentially due to poor structural understanding of the intricate interactions with their ligands. Indeed, about 50 chemokines and 20 receptors have been identified in humans. Their interactions require precise orchestration, as a chemokine may bind to several receptors, while a single chemokine receptor has multiple ligands. However, the structural determinants dictating ligand specificity and receptor activation remain elusive. Lately, cryo-EM has emerged as a ground-breaking technique for revealing molecular structures but despite recent advancements, analysis of small proteins like receptor:chemokine complexes (~50 kDa) is still challenging.
To address these difficulties and limitations, our ambition is to engineer a novel and generic tool to facilitate structural studies of chemokine receptors in complexes with their biological ligands using cryo-EM. For this purpose, we propose to generate chemokine-based chimeric proteins, called Megakines. To prove this concept and assess its applicability in structural biology, we chose to first reformat the well-characterized chemokine CCL5 and investigate its functionality towards two clinically relevant receptors, CCR5 and ACKR2.
Yet, although GPCRs are the target of about one third of currently marketed drugs, only three of them act on chemokine receptors, essentially due to poor structural understanding of the intricate interactions with their ligands. Indeed, about 50 chemokines and 20 receptors have been identified in humans. Their interactions require precise orchestration, as a chemokine may bind to several receptors, while a single chemokine receptor has multiple ligands. However, the structural determinants dictating ligand specificity and receptor activation remain elusive. Lately, cryo-EM has emerged as a ground-breaking technique for revealing molecular structures but despite recent advancements, analysis of small proteins like receptor:chemokine complexes (~50 kDa) is still challenging.
To address these difficulties and limitations, our ambition is to engineer a novel and generic tool to facilitate structural studies of chemokine receptors in complexes with their biological ligands using cryo-EM. For this purpose, we propose to generate chemokine-based chimeric proteins, called Megakines. To prove this concept and assess its applicability in structural biology, we chose to first reformat the well-characterized chemokine CCL5 and investigate its functionality towards two clinically relevant receptors, CCR5 and ACKR2.
As an alternative to crystallography, single-particle cryo-EM has become a versatile and ground-breaking technique for the structural analysis of macromolecular complexes. In single-particle cryo-EM, whereas large molecules (over 200 kDa) are relatively easily recognized in noisy low-dose images of frozen hydrated samples, it is much more difficult to collect and process images, and ultimately determine a 3D structure of small particles (below 100 kDa). The relatively small size of chemokines (~10 kDa), even in a complex with the interacting receptors (∼50 kDa), limits the eligibility of such important signaling complexes to benefit from the cryo-EM revolution with the currently available technology.
After an in-depth analysis of available structural information on chemokines and receptor:chemokine complexes, we proposed an innovative approach to increase the chemokine size, without altering its functionality. The fundamental design feature of such chimeras, called Megakines, is the insertion of a chemokine into a bulky well-structured scaffold protein via a rigid connection in order to build large, conformationally and functionally stable proteins. Accordingly, we first identified a region of a well-characterized chemokine CCL5 that does not directly interact with a receptor. Next, we inserted into this region genes encoding for circularly permutated large scaffold proteins such as HopQ or YgjK to generate 54 kDa and 92 kDa Megakines, respectively.
To experimentally validate the production of these novel chimeric proteins, and to identify the most favorable/functional connections between a chemokine and the two scaffold proteins, we designed a tailor-made in vitro selection method utilizing yeast display technology. In short, millions of chemokine-scaffold connection variants were displayed on yeast cells and simultaneously stained with a monoclonal antibody that binds only to a well-folded CCL5 chemokine, enabling selection of best-performing and functional Megakines.
The subsequent and instrumental aspect was to obtain large amounts of purified Megakine prototypes using the insect cell expression system, which is well established for production of active chemokines. Accordingly, several cHopQ- and cYgjK-based Megakines, together with CCL5 chemokine as a positive control, were expressed in Hi5 cells and subjected to a two-step purification procedure. As a result, we produced pure and stable protein samples in milligram quantitates, validated by various biophysical assays (SDS-PAGE, thermostability, mass spectrometry).
To validate their proper functionality and pharmacology, the purified samples were further characterized in dose-response fashion for their ability to activate the cognate receptors monitored through the induction of the recruitment of β-arrestin-1 to the receptors, in a close-to-native cellular environment. This state-of-the-art pharmacology validation approach demonstrated that all produced CCL5 Megakine variants can activate the cognate receptor, reaching the same efficacy as the parental CCL5 chemokine. However, the potencies were significantly reduced for enlarged CCL5 chemokines. We observed the same phenomenon for another enlarged chemokine, cHopQ-based CCL3 Megakine.
By definition, potency is a measure of ligand activity expressed in terms of the amount (concentration) required to yield an effect of given intensity (efficacy). Considering the receptor:Megakine interactions, we can conclude that Megakines can activate the cognate receptor, however, much higher concentrations are required to obtain the same activation/signaling levels than observed for native, unmodified chemokines. It directly corelates with a lower interaction strength, so-called affinity, between a Megakine and a receptor. This unforeseen observation can lead to an increased transient (low affinity) nature of the Megakine:receptor complexes, and thus obstructs their direct use is structural studies by single particle cryo-EM. In order to increase the affinity of this interaction and bypass observed limitations, development of novel and generic protein engineering approaches is ongoing as a part of further exploitation of this technology.
Dissemination of the Megakine project results is planned through a publication in a peer-reviewed international journal, under an open access format policy (gold or green). A manuscript describing the design, generation and pharmacology profiling of the first-in-class enlarged chemokines is in preparation.
After an in-depth analysis of available structural information on chemokines and receptor:chemokine complexes, we proposed an innovative approach to increase the chemokine size, without altering its functionality. The fundamental design feature of such chimeras, called Megakines, is the insertion of a chemokine into a bulky well-structured scaffold protein via a rigid connection in order to build large, conformationally and functionally stable proteins. Accordingly, we first identified a region of a well-characterized chemokine CCL5 that does not directly interact with a receptor. Next, we inserted into this region genes encoding for circularly permutated large scaffold proteins such as HopQ or YgjK to generate 54 kDa and 92 kDa Megakines, respectively.
To experimentally validate the production of these novel chimeric proteins, and to identify the most favorable/functional connections between a chemokine and the two scaffold proteins, we designed a tailor-made in vitro selection method utilizing yeast display technology. In short, millions of chemokine-scaffold connection variants were displayed on yeast cells and simultaneously stained with a monoclonal antibody that binds only to a well-folded CCL5 chemokine, enabling selection of best-performing and functional Megakines.
The subsequent and instrumental aspect was to obtain large amounts of purified Megakine prototypes using the insect cell expression system, which is well established for production of active chemokines. Accordingly, several cHopQ- and cYgjK-based Megakines, together with CCL5 chemokine as a positive control, were expressed in Hi5 cells and subjected to a two-step purification procedure. As a result, we produced pure and stable protein samples in milligram quantitates, validated by various biophysical assays (SDS-PAGE, thermostability, mass spectrometry).
To validate their proper functionality and pharmacology, the purified samples were further characterized in dose-response fashion for their ability to activate the cognate receptors monitored through the induction of the recruitment of β-arrestin-1 to the receptors, in a close-to-native cellular environment. This state-of-the-art pharmacology validation approach demonstrated that all produced CCL5 Megakine variants can activate the cognate receptor, reaching the same efficacy as the parental CCL5 chemokine. However, the potencies were significantly reduced for enlarged CCL5 chemokines. We observed the same phenomenon for another enlarged chemokine, cHopQ-based CCL3 Megakine.
By definition, potency is a measure of ligand activity expressed in terms of the amount (concentration) required to yield an effect of given intensity (efficacy). Considering the receptor:Megakine interactions, we can conclude that Megakines can activate the cognate receptor, however, much higher concentrations are required to obtain the same activation/signaling levels than observed for native, unmodified chemokines. It directly corelates with a lower interaction strength, so-called affinity, between a Megakine and a receptor. This unforeseen observation can lead to an increased transient (low affinity) nature of the Megakine:receptor complexes, and thus obstructs their direct use is structural studies by single particle cryo-EM. In order to increase the affinity of this interaction and bypass observed limitations, development of novel and generic protein engineering approaches is ongoing as a part of further exploitation of this technology.
Dissemination of the Megakine project results is planned through a publication in a peer-reviewed international journal, under an open access format policy (gold or green). A manuscript describing the design, generation and pharmacology profiling of the first-in-class enlarged chemokines is in preparation.
There is a constant high demand in the field of drug discovery for a better understanding of the chemokine receptor biology, especially at the structural level. Indeed, only in last two years, a number of novel structures of receptor:chemokine complexes were solved by cryo-EM, one of which, CCR6:CCL20 in particular, was described by Pfizer. The high-resolution reconstructions of all of these structures were strongly enabled by the receptor interaction with a G protein and an antibody fragment, stressing the necessity to increase the size of the complex to make it eligible for cryo-EM analysis. We still recognise a strong possible applicability of our unique solution for a routine analysis of different receptor:chemokine complexes, however, the interaction affinity between Megakine and a cognate receptor needs to be further improved.