Small Molecule Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Inhibitors: Hit to Lead Optimization of Systemic Agents

Abstract

The optimization of a new class of small molecule PCSK9 mRNA translation inhibitors is described. The potency, physicochemical properties, and off-target pharmacology associated with the hit compound (1) were improved by changes to two regions of the molecule. The last step in the synthesis of the congested amide center was enabled by three different routes. Subtle structural changes yielded significant changes in pharmacology and off-target margins. These efforts led to the identification of 7l and 7n with overall profiles suitable for in vivo evaluation. In a 14-day toxicology study, 7l demonstrated an improved safety profile vs lead 7f. We hypothesize that the improved safety profile is related to diminished binding of 7l to nontranslating ribosomes and an apparent improvement in transcript selectivity due to the lower strength of 7l stalling of off-target proteins.

Cellular response to small molecules that selectively stall protein synthesis by the ribosome

“…We also compared our present PF846 results with the 21 PF846 targets previously identified in Huh-7 cells (S3D Fig and S2 Table) and observed that changes in the read count 3' of the stall site defined by DMax were highly correlated between experiments (Pearson R=0.82), showing the high reproducibility of our method of analysis (S2 Fig). However, we found that the main stall site position seen in the ribosome profiling read-counts varied slightly when comparing the present PF846 Liaud et al 9 treatment and the previously published PF846 data [10], and when comparing PF8503 and PF846 stall sites on common targets (S4 Fig, S1 Table and S2 Table). This could be due to technical variability in the preparation of the ribosome profiling libraries, such as the efficiency of RNAse I digestion.…”

“…The same experiment and pipeline was used in parallel with PF846 in order to compare our results to previously published data [10]. After using the bioinformatic pipeline described previously [9] (S2A Fig), we found that PF8503 affected the translation of 46 mRNAs, whereas PF846 affected translation of 24 mRNAs after 1 hr of Liaud et al 8 treatment. As expected PCSK9 was affected by both compounds with a log 2 fold change of -1.5 and -2 for PF8503 and PF846, respectively (Fig 1C and S1 Table).…”

“…Previous studies showed that cells from the hematopoietic lineage are more sensitive to the class of compounds that include PF846 and PF8503 (Fig 1A) [9]. In these experiments, which used rat bone marrow, PF846 and PF8503 had similar toxicity profiles [9]. The CRISPRi screen library was originally developed and validated in K562 cells [15], immortalized leukemia cells that Liaud et al 7 can be induced to develop characteristics similar to early-stage erythrocytes, granulocytes and monocytes [18].…”

“…Here, we used a genomic CRISPRi screen using a compound that selectively stalls the translation of human PCSK9, to test the effects of this class of compound on human cell fitness. We used compound PF-06378503 (PF8503) [9], a compound related to the PCSK9 selective translational inhibitor PF-06446846 (PF846) described previously [10], but which is slightly more toxic, to exert the selective pressure required for growth-based CRISPRi screens [17]. We show using ribosome profiling that PF8503 inhibits Liaud et al 6 translation of an overlapping set of proteins compared to PF846, along with a distinct set of off-target proteins.…”

“…As opposed to affecting translation for much of the proteome, a new class of compounds has been identified that selectively targets the translation of PCSK9, with few off-target effects [9][10][11]. These compounds bind in the ribosome exit tunnel [12], Liaud et al 5 and seem to affect the trajectory of the growing nascent polypeptide chain in the exit tunnel as it is extended, thereby selectively stalling translation of a narrow spectrum of transcripts.…”