A therapeutic target for ischemic related injuries, application to organ transplantation – KIRI

Task 1.1: organic synthesis of 5'-N-L-seryl-sulfamoyl adenosine (SFA)
SFA was synthetized following a modified protocol from the classical organic synthesis method already described. We prepare various analogs upon modification of the linker using carbamate, ester and triazole analogues as more stable alternatives for SFA derivatives
Task 1.2 Biological activity of the SFA and of the synthesized analogs
The serRS inhibitors obtained during Task 1.1 is screened for their ability to inhibit the translation of the luciferase reporter protein using an in vitro synthesis protocol. This screening gives a dose-response inhibition curve. The second screening is performed on renal cultured cells submitted to anoxia (cell death and oxidative stress measurement)
Task 1.3 Measurement of cell metabolism parameters
In parallel to the determination of the biological activity of the serRS inhibitors, it is important to analyze some classical parameters. To this aim, we check the main pathway allowing ischemia resistance and ATP preservation in our model, as protein synthesis inhibition, bioenergetic profile or mitochondria phenotype. The cell protein content in each experimental condition is quantified using a colorimetric assay kit. Measurement of cellular ATP content is performed using a luciferin/luciferase assay kit. The bioenergetic profiles is established using a Seahorse XF96 Extracellular Flux.
Task 2: Chemical synthesis of new serine derivatives and evaluation of their biological activity
Task 2.1 Chemical synthesis of new SFA derivatives
In order to increase the affinity for the target as well as to maintain the selectivity of the initial inhibitors SFA we design new analogs that include modifications of both the seryl moiety (amino acid side chain) and the adenine nucleobase. First, we perform a molecular docking study using the reported crystal structure of serRS. We design new inhibitors bearing potentially an improved activity as well as physico-chemical properties favorable for an eventual therapeutic application. We modify the serine moiety and the nucleobase moiety. The purity of the obtained derivatives is assessed by classical analytical methods such as HPLC-MS analysis and NMR characterization.
Task 2.2 The biological activity of the newly synthesized compounds will be confirmed upon studying their activities on in vitro protein translation and in cultured cells submitted to anoxia. This task is similar to task 1.2 concerning the previous serRS inhibitors.
Task 2.3 Physiological application of the most relevant serine derivatives on ischemia/reperfusion injury in rat kidney.
Rats are treated or not with the inhibitors (concentrations defined in Task 2,2). Unilateral renal artery ischemia (40 min.) is performed and renal function is assessed in the following 24h. The early biomarker of kidney injury NGAL is measured together with the main tubular transport functions (fractional excretion of glucose, sodium, phosphate).

- In accordance with the project we synthesized 5'-N-L-seryl-sulfamoyl adenosine (SFA) according to the protocol that we have developed

- We were able to show that this inhibitor was capable of inhibiting seryl tRNA synthetase using an in vitro translation technique from rabbit reticulocytes and the reporter protein luciferase. We were able to determine its IC50 in vitro which is around the micromole.


- We were able to show that the synthesized SFA possessed a protective action against anoxia on renal cells in culture and on neurons in primary cultures. The concept of inhibition of serRS as a protective effect against an ischemic episode therefore seems validated.


- The first part of the project also concerns the synthesis of new stable analogues of 5'-N-L-seryl-sulfamoyl adenosine by modification of the linker between the serine and adenosine fragments. Unfortunately, this task started later than expected due to the SARS-CoV-2 pandemic which prevented Partner 2 from recruiting on time the required non-permanent engineer to perform the synthesis. The preparation of the new analogues started only a few months ago with the synthesis of two triazole analogues. These compounds have never been described in the literature and we had to set up the conditions for each step of their chemical synthesis. The preparation of each compound requires 6 steps. The compounds are under characterization and will soon be available for their biological evaluation.


5'-O-seryltriazolyladeonsin A 5'-O-seryltriazolyladeonsine B
triazole analogs of SFA being synthesized.

- From a biological point of view, as planned, we analyzed the effects of inhibition of the protection against ischemia pathway on metabolic parameters. The mechanism demonstrated involves a metabolic change from oxidative phosphorylation to glycolysis allowing kidney cells to be transiently independent of oxygen supply. We have shown that GC7 decreases the expression of renal glucose transporter GLUT1 proteins leading to a decrease in trans-cellular glucose flux. At the same time, GC7 changes the native energy source of proximal cells from glutamine to glucose utilization. Thus, GC7 acutely and reversibly reprograms the function and metabolism of kidney cells to make glucose its sole substrate, thus allowing cells to be independent of oxygen through anaerobic glycolysis. The physiological consequences are an increase in renal excretion of glucose and lactate, reflecting a decrease in glucose reabsorption and an increase in glycolysis.

Remarkable results:
- The serRS protein is a pharmacological target against renal and neuronal ischemia
- Inhibition of the eIF5A pathway reprograms renal glucose metabolism

The originality of this project in the cell biology, physiology and clinical fields is based on a new paradigm that goes beyond the current knowledge in the field of ischemia and that paves the way for a new field of investigation in that subject. The serRS involvement together with the eIF5A molecular pathway was unexpected and the discovery of original and efficient inhibitors of serRS will pave the way for an accurate pharmacological management of ischemic-reperfusion and also stroke injuries for which and up to now no drugs have been successfully used in human health. Our two successive registered patents related to ischemic tolerance have acknowledged this breakthrough (1: WO2012010641 pharmaceutical composition for increasing cellular hypoxic tolerance. 2012; 2: EP18176120 Modulators and pharmaceutical compositions comprising the same for increasing cell hypoxic tolerance. 2018).
Besides the discovery of a validated very new target/drug pair usable in human ischemic pathologies this work aims to progress in the fundamental knowledge of the ischemic and hypoxic related cellular network that is currently poorly understood. Identification in the future of a new generation of targets requires a thorough understanding of the processes driving ischemic tolerance. The goal is not only to validate the present concept in animal models of human pathologies but also mechanisms controlling this new pathway that could be therapeutics opportunities for the next generation of ischemic tolerance related drugs.

Inhibition of eIF5A hypusination reprogrammes metabolism and glucose handling in mouse kidney.
Cougnon M, Carcy R, Melis N, Rubera I, Duranton C, Dumas K, Tanti JF, Pons C, Soubeiran N, Shkreli M, Hauet T, Pellerin L, Giraud S, Blondeau N, Tauc M, Pisani DF.
Cell Death Dis. 2021 Mar 17;12(4):283. doi: 10.1038/s41419-021-03577-z.
Targeting oxidative stress, a crucial challenge in renal transplantation outcome.
Carcy R, Cougnon M, Poet M, Durandy M, Sicard A, Counillon L, Blondeau N, Hauet T, Tauc M, F Pisani D.
Free Radic Biol Med. 2021 Jun;169:258-270. doi: 10.1016/j.freeradbiomed.2021.04.023. Epub 2021 Apr 21.

This project aims to design and physiologically use a new generation of pharmacological inhibitors targeting the ischemic stress through a protein we have recently identified as implicated in the resistance of tissues and organs to ischemia. The pre-clinical application that is concerned in the project is kidney transplantation.
In human health the prevention of the consequences of an organic ischemic stress, due to oxygen deprivation, is one of the major concerns of clinicians. This stress occurs in pathological situations such as stroke, myocardial infarction, aortic surgery or organ transplantation. In the latter case, the ischemic stress is scheduled by the clinician who can therefore apply pre-conditioning protocols able to prepare the organ to resist against this stress. Unfortunately clinicians are lacking treatment against these ischemic pathologies due to the absence of identified pharmacological targets. A study conducted by Vigne et al. (2008) opened a new field of investigation by highlighting in Drosophila a new paradigm in tolerance to hypoxia. These authors have shown the primordial role of polyamines in oxygen sensitivity and they have identified the eukaryotic initiation factor 5A (eIF5A) and more particularly its activation step as being a pharmacological target of ischemic tolerance. We have transposed this new concept in mammals and have shown that the inhibition of eIF5A activation by GC7 (N-guanyl-1,7-diaminoheptane) protects renal function from an ischemic stress induced by temporary occlusion of the renal artery in rodent. This protective effect is consecutive to a decrease in oxidative phosphorylation activity associated to a decrease in oxidative stress. This pharmacological method of increasing ischemic tolerance via the targeting of the eIF5A hypusination step has been recently protected by an international patent (WO / 2012/010641). Applied to kidney transplantation at the pre-clinical level in pigs we have also shown that the pre-conditioning of the donor with GC7 allows a very clear improvement in the recovery of renal function of the recipient. eIF5A is an important element in the protein translation machinery and controls the specific synthesis of numerous proteins. To identify the primary target implicated in ischemic tolerance we conducted a differential proteomic study associated with mass spectrometry that allowed us to identify a candidate protein whose identity will not be revealed because of patent pending. Gene inhibition of this protein by siRNA or low affinity inhibitors has confirmed its role in ischemic tolerance either in vitro or in vivo.
Our goal is to engineer, by organic synthesis, high affinity inhibitors for this protein target and, once characterized in vitro, to use them in vivo in a preclinical model of renal transplantation in pigs to ultimately test them in clinical trials.
The first trials led with an home made specific inhibitor used for crystallography experiments of this protein in the nineties gave a positive result on the increase in anoxic tolerance in vitro confirming the relevance of the concept.
On this project we have gathered a consortium of 3 laboratories with the necessary complementary expertise. UMR7370 Nice, inventor of the concept, expert in cell biology, renal physiology and oxidative stress management; UMR7272 (Nice Institute of Chemistry) specialized in organic synthesis of bio-active molecules; U1082 (University Hospital of Poitiers), specialist in renal transplantation at a preclinical level in pigs. We ask the ANR support 500k € for the three teams and for 3 years. In the future the development of this concept could apply to all pathological or clinical areas in which ischemic stress is involved.