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Disease related mutations in PI3Kγ disrupt regulatory C-terminal dynamics and reveals a path to selective inhibitors

Class I Phosphoinositide 3-kinases (PI3Ks) are master regulators of cellular functions, with the p110γ subunit playing a key role in immune signalling. PI3Kγ is a key factor in inflammatory diseases, and has been identified as a therapeutic target for cancers due to its immunomodulatory role. Using a combined biochemical/biophysical approach, we have revealed insight into regulation of kinase activity, specifically defining how immunodeficiency and oncogenic mutations of R1021 in the c-terminus can inactivate or activate enzyme activity. Screening of small molecule inhibitors using HDX-MS revealed that activation loop binding inhibitors induce allosteric conformational changes that mimic those seen for the R1021C mutant. Structural analysis of clinically advanced PI3K inhibitors revealed novel binding pockets that can be exploited for further therapeutic development. Overall this work provides unique insight into the regulatory mechanisms that control PI3Kγ kinase activity, and shows a framework for the design of PI3K isoform and mutant selective inhibitors.


Abstract Introduction 38
The phosphoinositide 3-kinase (PI3K) family of enzymes are central regulators of 39 growth, proliferation, migration, and metabolism in a plethora of cells and tissues [1,2]. 40 PI3Ks are lipid kinases that generate the lipid second messenger phosphatidylinositol 41 3,4,5 trisphosphate (PIP3), which is utilised downstream of cell surface receptors to 42 regulate growth, metabolism, survival, and differentiation [1]. PIP3, is generated by four 43 distinct class I PI3K catalytic isoforms separated into two groups (class IA [p110a, p110b, 44 p110d], and class IB [p110g] (sometimes referred to as PI3Ka, PI3Kb, PI3Kd, and PI3Kg 45 catalytic subunit)). The primary difference between class IA and class IB PI3Ks is their 46 association with specific regulatory subunits, with class IA binding p85-like regulatory 47 subunits encoded by PIK3R1, PIK3R2, PIK3R3, and PI3Kg forming complexes with either 48 a p101 or p84 (also called p87 PIKAP ) adaptor subunit [3][4][5]. The four isoforms of class I 49 PI3K have distinct expression profiles, with PI3Ka and PI3Kb being ubiquitously 50 expressed, and PI3Kd and PI3Kg being mainly localised in immune cells [1]. All PI3K 51 isoforms have been implicated in a variety of human diseases, including cancer, 52 immunodeficiencies, inflammation, and developmental disorders [6][7][8]. 53 The class IB PI3Kg isoform encoded by PIK3CG is a master regulator of immune 54 cell function. It plays important roles in the regulation of myeloid (macrophages, mast 55 cells, neutrophils) and lymphoid (T cells, B cells, and Natural Killer cells) derived immune 56 cells [9][10][11]. PI3Kg regulates immune cell chemotaxis [11][12][13], cytokine release [14,15], 57 and generation of reactive oxygen species [11], which are important processes in both the 58 innate and adaptive immune systems. The ability of PI3Kg to mediate multiple immune 59 cell functions is controlled by its activation downstream of numerous cell surface 60 receptors, including G-protein coupled receptors (GPCRs) [16], the IgE/Antigen 61 receptor [14], receptor tyrosine kinases (RTKs) [17], and the Toll-like receptors (TLRs) 62 [18,19]. Activation of PI3Kg downstream of these stimuli are potentiated by their p84 and 63 p101 regulatory subunits [5,18,[20][21][22]. This is distinct from the roles of regulatory subunits 64 in class IA PI3Ks, which act as potent inhibitors of p110 catalytic activity [23]. In mouse 65 models, loss of PI3Kg either genetically or pharmacologically is protective in multiple 66 isoforms, this inhibited conformation requires interactions with a p85 regulatory subunit 89 ( Fig. S1) [8]. In PI3Kg this inhibited conformation is proposed to be maintained by a 90 Tryptophan lock, where W1080 maintains a closed conformation of the membrane 91 binding C-terminal ka12 helix, leading to an inactive conformation of the activation loop 92 [34] (Fig. 1B+C).       E. Activity assays testing the intrinsic ATPase activity (ATP conversion in the absence of membrane 164 substrate) for wild type and mutant p110g/p101 complexes.

165
F. Lipid kinase activity assays testing the activity of WT and R1021C for the free p110g catalytic subunit 166 with and without lipidated Gbg. Lipid kinase activity was generated by subtracting away non-specific ATPase 167 activity, for unstimulated WT p110g there was no detectable lipid kinase activity above basal ATPase activity 168 (N.D.). For panels D-F, every replicate is plotted, with error shown as standard deviation (n=3-6). Two tailed 169 p-values represented by the symbols as follows: ***<0.001; **<0.01; *<0.05; N.S.>0.05.

171
The R1021 residue forms hydrogen bonds with the carbonyl oxygens of L1055, 172 T1056, and K1059 located in or adjacent to the regulatory arch helices ka10 and ka11 of 173 PI3Kg (Fig 1C). Both R1021C and R1021P would be expected to disrupt these 174 interactions, with the R1021P also expected to distort the secondary structure of the ka8 175 helix. The R1021P has been previously found to lead to greatly decreased lipid kinase 176 activity in vitro [46]. To characterize these mutations, we carried out biochemical assays 177 of wild-type, R1021C, and R1021P p110g/p101 complexes against plasma membrane-178 mimic lipid vesicles containing 5% PIP2. Assays were carried out in the presence and 179 absence of lipidated Gβg subunits, a potent p110g/p101 activator. These assays revealed 180 that p110g/p101 R1021C was ~8-fold more active than wild-type both basally and in the 181 presence of Gβg (Fig. 1D). The R1021P complex showed greatly decreased Gβg 182 stimulation compared to wild-type. Intriguingly, R1021P showed higher basal ATPase 183 activity (non-productive turnover of ATP) compared to WT, revealing that it still has 184 catalytic activity, but greatly decreased activity on lipid substrate (Fig. 1E). The R1021C 185 mutant also showed a ~8-fold increase in lipid kinase activity compared to wild-type when 186 assaying the free 110g subunit (Fig. 1F) (Table S1).    To obtain further insights into the dynamic behavior of the C-terminus of the kinase 281 domain and how mutation of R1021 alters conformational dynamics, we monitored the 282 fluctuations of four different angles formed between ka8, ka9, ka10, and ka11 (Fig. 3E). 283 The simulations revealed increased angle fluctuations in the mutant simulations between 284 ka8 and ka9, and ka9 and ka10, with a bimodal distribution in the ka8/ka9 angle 285 compared to WT. There was also increased fluctuations in the activation loop in the 286 mutants compared to WT ( Figure 3C, Fig. S4).       Deuterium exchange experiments were carried out with monomeric p110g over 4 313 timepoints of deuterium exchange (3,30,300, and 3000 s). We obtained 180 peptides 314 covering ~89% percent of the exchangeable amides (Table S1). To verify that results on 315 the free p110g complex are relevant to the physiological p110g/p101 complex, we also 316 carried out experiments with the p110g/p101 complex with Gedatolisib and IPI-549, with 317 the free p110g showing almost exactly the same differences as seen for the p110g/p101 318 complex (Fig. S5). 319 320     This shows that multiple PI3K inhibitors can cause large scale allosteric 361 conformational changes upon inhibitor binding, however, deciphering the molecular 362 mechanism of these changes were hindered by lack of high-resolution structural 363 information for many of these compounds. 364 365

Structures of PI3Kg bound to IPI-549, Gedatolisib, and NVS-PI3-4 366
To further define the molecular basis for how different inhibitors lead to allosteric 367 conformational changes we solved the crystal structure of p110g bound to IPI-549, 368 Gedatolisib, and NVS-PI3-4 at resolutions of 2.55Å, 2.65Å, and 3.15Å, respectively ( Idelalisib bound to p110d revealed a potential molecular mechanism for p110g selectivity.

405
One of the most striking differences between the structure of Gedatolisib and IPI-406 549 bound to p110g is the conformation of the N-terminus of the activation loop, including 407 the residues that make up the DFG motif (Fig. 5B, D+E of the cyclopropyl motif in AZ2 with the activation loop has previously been proposed to 412 be critical in mediating allosteric conformational changes. In addition to the change in the 413 activation loop, there was a minor perturbation of the W1080 lock, with the Gedatolisib 414 structure revealing a disruption of the hydrogen bond between W1080 and D904, with 415 this bond maintained in the IPI-549 structure (Fig. 5F+G). The C-terminus of the activation 416 loop and ka7 immediately following showed some of the largest changes upon inhibitor 417 binding in HDX experiments. The ka7 helix is directly in contact with W1080, and we 418 postulated that the conformational changes induced in the N-terminus of the activation 419 loop may mediate these changes. 420  We observed that HDX differences occurring in the R1021C mutant, were very 433 similar to conformational changes observed for p110g bound to Gedatolisib, particularly 434 for the peptide spanning 976-992 in the activation loop. As this region is directly adjacent 435 to the inhibitor binding site, we postulated that there may be altered inhibitor binding for 436 the R1021C mutant. We carried out IC50 measurement for wild-type and R1021C 437 p110g/p101 with both IPI-549 and Gedatolisib (Fig. 6A). Gedatolisib was roughly three-438 fold more potent for the R1021C mutant over the wild-type, with no significant difference 439 in IC50 values for IPI-549 compared to wild-type. This provides initial insight into how 440 understanding the dynamics of activating mutations and inhibitors may be useful as a 441 novel strategy towards designing mutant specific inhibitors. The extruded vesicles were sonicated again for 5 minutes, aliquoted and stored at -80°C. 659 For inhibitor response assays, lipid vesicles containing 95% PS and 5% C8-PIP2 were 660 used. PS was dried and desiccated as described above. The lipid film was mixed and 661 resuspended with C8-PIP2 solution (2.5 mg/mL in lipid buffer). Following this, vesicles 662 were essentially prepared the same way as described above. All vesicles were stored at 663 5 mg/mL. of 2X protein solution to start the reaction and allowed to proceed for 60 minutes at 37 o C. 688 Following this, the reaction was stopped and the intensity was measured. The raw data 689 was normalized against a 0-100% ADP window as described above. The % inhibition was 690 calculated by comparison to the activity with no inhibitor to obtain fraction activity 691 remaining. The generated models were prepared using tleap program of the AMBER package [92]. 778 The systems were parametrized using the general AMBER force field (GAFF) using 779 cycles. During the first cycle, the protein was restrained using a harmonic potential with 789 a force constant of 10 kcal/mol-Å 2 , while the solvent, and ions were subjected to an initial 790 minimization of 2000 steps using the steepest descent approach for 1000 steps and 791 conjugate gradient approach for another 1000 steps. The full system (protein + solvent) 792 was then similarly minimized for 1000 and 4000 steps using the steepest descent and 793 conjugate gradient approaches, respectively. Subsequently, the temperature was 794 incrementally changed from 100 to 300 K for 10 ps at 2 fs/step (NVT ensemble). Next,         Omipalisib bound to p110g were from PDB: 2CHX [8] and 3l54 [9], respectively.