(1105-B) IMMUNATURAL: An HTS Platform for the discovery of small-molecules from microbial origin against the PD-1/PD-L1 protein-protein interaction.
Monday, February 5, 2024
2:00 PM – 3:00 PM EST
Location: Exhibit Halls AB
Abstract: The PD-1/PD-L1 protein-protein interaction (PPI) controls an adaptive immune resistance mechanism exerted by tumor cells in response to endogenous immune anti-tumor activity. The large-molecule nature of current commercial monoclonal antibodies (mAbs) against this interaction makes them expensive treatments, and in many cases ineffective due to their limited bioavailability and their propensity to produce severe side effects. However, current PD-1/PD-L1 small-molecule inhibitors are of synthetic origin and have not passed clinical trials. To fill this gap, we have developed the first high throughput screening (HTS) cellular assay that is suitable for the identification of bioactive, potent, and specific PD-1/PD-L1 inhibitors from microbial natural product (NP) libraries of maximized taxonomical and biosynthetic diversity, such a structurally diverse chemical space with minimum overlap with current synthetics. The HTS assay was developed using miniaturized 384-wells plates and automated/acoustic liquid handling devices. For a pilot screening, we screened 6000 microbial extracts from 1500 fungal and bacterial strains. The resulting screening met the quality standards of the HTS format (Z´-factor >0.5, assay window >5) and followed a bimodal distribution in which the normal population of active extracts and positive controls presented z-scores statistically different from the normal population of inactive extracts and negative controls, altogether demonstrating the robustness and scalability of our new HTS platform. Confirmed hits (hit rate 0.3 %) were subjected to LC-MS analysis (de-replication) to detect already known NPs potentially responsible for the PD-1/PD-L1 inhibitory activity that would validate our platform. In one of the bioactive extracts containing known NPs, we detected a fungal small-molecule, which was further isolated in its pure form and confirmed as PD-1/PD-L1 inhibitor from the crude extract of the refermented strain. The corresponding low micromolar IC50 value in the cellular PD-1/PD-L1 blockade assay followed a similar potency trend in a PD-1/PD-L1 cell-free AlphaLISA assay and orthogonal biophysical assays reporting for PD-1 and PD-L1 specificity. Thus, our fungal NP is a first-in-class inhibitor that demonstrates the unprecedented efficiency of our HTS cellular setup at capturing specific PD-1/PD-L1 inhibitors from chemically diverse libraries of NPs microbial extracts. Further efforts are ongoing on a different subset of highly potent extracts from where completely novel NPs/molecular formulas have been postulated as PD-1/PD-L1 inhibitors. 1) The final isolation and structural elucidation of such new molecular formulas and 2) the identification of nanomolar analogues of our first-in-class fungal PD-1/PD-L1 inhibitor from scanning a variety of fermentation conditions, and biosynthetically related strains, are two ongoing lines of research. From these new lines of research, we expect to obtain more rock-solid proofs to demystifying NP drug discovery as a long/misleading process, but rather as an unexploited source of new chemical structures with immunotherapeutic properties so far unattainable by means of synthetic chemistry or repurposing commercial libraries.