PF-07059013: A Noncovalent Modulator of Hemoglobin for Treatment of Sickle Cell Disease.

Sickle cell disease (SCD) is a genetic disorder caused by a single point mutation (ß6 Glu → Val) on the ß-chain of adult hemoglobin (HbA) that results in sickled hemoglobin (HbS). In the deoxygenated state, polymerization of HbS leads to sickling of red blood cells (RBC). Several downstream consequences of polymerization and RBC sickling include vaso-occlusion, hemolytic anemia, and stroke. We report the design of a noncovalent modulator of HbS, clinical candidate PF-07059013 (23). The seminal hit molecule was discovered by virtual screening and confirmed through a series of biochemical and biophysical studies. After a significant optimization effort, we arrived at 23, a compound that specifically binds to Hb with nanomolar affinity and displays strong partitioning into RBCs. In a 2-week multiple dose study using Townes SCD mice, 23 showed a 37.8% (±9.0%) reduction in sickling compared to vehicle treated mice. 23 (PF-07059013) has advanced to phase 1 clinical trials.

The catalytic mechanism of cyclic GMP-AMP synthase (cGAS) and implications for innate immunity and inhibition.

Cyclic GMP-AMP synthase (cGAS) is activated by ds-DNA binding to produce the secondary messenger 2',3'-cGAMP. cGAS is an important control point in the innate immune response; dysregulation of the cGAS pathway is linked to autoimmune diseases while targeted stimulation may be of benefit in immunoncology. We report here the structure of cGAS with dinucleotides and small molecule inhibitors, and kinetic studies of the cGAS mechanism. Our structural work supports the understanding of how ds-DNA activates cGAS, suggesting a site for small molecule binders that may cause cGAS activation at physiological ATP concentrations, and an apparent hotspot for inhibitor binding. Mechanistic studies of cGAS provide the first kinetic constants for 2',3'-cGAMP formation, and interestingly, describe a catalytic mechanism where 2',3'-cGAMP may be a minor product of cGAS compared with linear nucleotides.

Binding screen for cystic fibrosis transmembrane conductance regulator correctors finds new chemical matter and yields insights into cystic fibrosis therapeutic strategy.

The most common mutation in cystic fibrosis (CF) patients is deletion of F508 (ΔF508) in the first nucleotide binding domain (NBD1) of the CF transmembrane conductance regulator (CFTR). ΔF508 causes a decrease in the trafficking of CFTR to the cell surface and reduces the thermal stability of isolated NBD1; it is well established that both of these effects can be rescued by additional revertant mutations in NBD1. The current paradigm in CF small molecule drug discovery is that, like revertant mutations, a path may exist to ΔF508 CFTR correction through a small molecule chaperone binding to NBD1. We, therefore, set out to find small molecule binders of NBD1 and test whether it is possible to develop these molecules into potent binders that increase CFTR trafficking in CF-patient-derived human bronchial epithelial cells. Several fragments were identified that bind NBD1 at either the CFFT-001 site or the BIA site. However, repeated attempts to improve the affinity of these fragments resulted in only modest gains. Although these results cannot prove that there is no possibility of finding a high-affinity small molecule binder of NBD1, they are discouraging and lead us to hypothesize that the nature of these two binding sites, and isolated NBD1 itself, may not contain the features needed to build high-affinity interactions. Future work in this area may, therefore, require constructs including other domains of CFTR in addition to NBD1, if high-affinity small molecule binding is to be achieved.