Exploiting transient protein states for the design of small-molecule stabilizers of mutant p53

The destabilizing p53 cancer mutation Y220C creates an extended crevice on the surface of the protein that can be targeted by small-molecule stabilizers. Here, we identify different classes of small molecules that bind to this crevice and determine their binding modes by X-ray crystallography. These structures reveal two major conformational states of the pocket and a cryptic, transiently open hydrophobic subpocket that is modulated by Cys220. In one instance, specifically targeting this transient protein state by a pyrrole moiety resulted in a 40-fold increase in binding affinity. Molecular dynamics simulations showed that both open and closed states of this subsite were populated at comparable frequencies along the trajectories. Our data extend the framework for the design of high-affinity Y220C mutant binders for use in personalized anticancer therapy and, more generally, highlight the importance of implementing protein dynamics and hydration patterns in the drug-discovery process

Small molecule induced reactivation of mutant p53 in cancer cells

The p53 cancer mutant Y220C is an excellent paradigm for rescuing the function of conformationally unstable p53 mutants because it has a unique surface crevice that can be targeted by small-molecule stabilizers. Here, we have identified a compound, PK7088, which is active in vitro: PK7088 bound to the mutant with a dissociation constant of 140 μM and raised its melting temperature, and we have determined the binding mode of a close structural analogue by X-ray crystallography. We showed that PK7088 is biologically active in cancer cells carrying the Y220C mutant by a battery of tests. PK7088 increased the amount of folded mutant protein with wild-type conformation, as monitored by immunofluorescence, and restored its transcriptional functions. It induced p53-Y220C-dependent growth inhibition, cell-cycle arrest and apoptosis. Most notably, PK7088 increased the expression levels of p21 and the proapoptotic NOXA protein. PK7088 worked synergistically with Nutlin-3 on up-regulating p21 expression, whereas Nutlin-3 on its own had no effect, consistent with its mechanism of action. PK7088 also restored non-transcriptional apoptotic functions of p53 by triggering nuclear export of BAX to the mitochondria. We suggest a set of criteria for assigning activation of p53

Mutations in sphingosine-1-phosphate lyase cause nephrosis with ichthyosis and adrenal insufficiency

Steroid-resistant nephrotic syndrome (SRNS) causes 15% of chronic kidney disease cases. A mutation in 1 of over 40 monogenic genes can be detected in approximately 30% of individuals with SRNS whose symptoms manifest before 25 years of age. However, in many patients, the genetic etiology remains unknown. Here, we have performed whole exome sequencing to identify recessive causes of SRNS. In 7 families with SRNS and facultative ichthyosis, adrenal insufficiency, immunodeficiency, and neurological defects, we identified 9 different recessive mutations in SGPL1, which encodes sphingosine-1-phosphate (S1P) lyase. All mutations resulted in reduced or absent SGPL1 protein and/or enzyme activity. Overexpression of cDNA representing SGPL1 mutations resulted in subcellular mislocalization of SGPL1. Furthermore, expression of WT human SGPL1 rescued growth of SGPL1-deficient dpl1. yeast strains, whereas expression of disease-associated variants did not. Immunofluorescence revealed SGPL1 expression in mouse podocytes and mesangial cells. Knockdown of Sgpl1 in rat mesangial cells inhibited cell migration, which was partially rescued by VPC23109, an S1P receptor antagonist. In Drosophila, Sply mutants, which lack SGPL1, displayed a phenotype reminiscent of nephrotic syndrome in nephrocytes. WT Sply, but not the disease-associated variants, rescued this phenotype. Together, these results indicate that SGPL1 mutations cause a syndromic form of SRNS

SAMPL6: pKa prediction blind challenge submission

In a collaboration with Prof. Stefan Grimme (Bonn University, Germany) we will submit predicted pKa (dissociation constant) values for the pKa prediction part of the SAMPL6 challenge (https://drugdesigndata.org/about/sampl6/pka-prediction). The SAMPL6 challenge is a public challenge where participants are invited to submit predictions of microscopic and macroscopic pKa values of 24 small molecules. The structures of the small molecules are in the public domain and can be found here: https://github.com/MobleyLab/SAMPL6/blob/pKa/images/pKa_challenge_small_molecules.jpg . We will apply the conformational workflow ReSCoSS (previously externally presented as OAK IDs 32054 and 34000) to this challenge. All molecules treated are in the public domain and the tools used have all been previously published. In line with the concept of the SAMPL6 challenge, we plan to submit the predicted values to the SAMPL6 challenge organizers, and for generating them we are collaborating with Prof. Grimme's group

Manuscript: SGPL1 mutations cause nephrosis with ichthyosis and adrenal insufficiency. Note: Details of NIBR contribution to manuscript: The results of Mutational analysis of SGPL1: In silico modelling ( were approved in OAK before-ref link https://oak-intra.novartis.com/25460/). Mutational analysis of SGPL1: In silico modelling results were shared with Prof. Friedhelm Hildebrandt, Harvard medical school, Division of Nephrology Boston Children's Hospital. These results, that were shared with Prof. Friedhelm Hildebrandt is now a part of full manuscript for publication with Prof. Friedhelm Hildebrandt as corresponding author . Honnappa Srinivas and Rainer Wilcken are the co-authors from NIBR. Please find attached the full manuscript.

ABSTRACT Steroid-resistant nephrotic syndrome (SRNS) causes 15% of chronic kidney disease. A mutation in one of >40 different monogenic genes can be detected in ~30% of individuals with SRNS who manifest before 25 years of age. However, in many patients the genetic etiology remains unknown. We performed whole exome sequencing (WES) to identify novel recessive causes of SRNS. In 7 families with a new syndrome of SRNS and facultative ichthyosis, adrenal insufficiency, immunodeficiency and neurological defects, we identified 9 different recessive mutations in SGPL1 encoding sphingosine-1-phosphate lyase. All mutations resulted in reduced or absent SGPL1 protein and/or enzyme activity. Overexpression of cDNA representing mutations of SRNS patients resulted in subcellular mislocalization of SGPL1. Furthermore, expression of WT human SGPL1 rescued growth of SGPL1-deficient dpl1 yeast strains, whereas expression of disease-associated variant proteins did not. Immunofluorescence revealed SGPL1 expression in mouse podocytes and mesangial cells. Knockdown of SGPL1 in rat mesangial cells (RMC) inhibited cell migration, which was partially rescued by VPC23109, an S1P receptor antagonist. Knockdown of SGPL1 in RMC resulted in a decrease of active RAC1 and CDC42, consistent with previously noted podocytic imbalance of active RAC1 in the pathogenesis of SRNS. In Drosophila Sply mutants, which lack SGPL1, a phenotype reminiscent of nephrotic syndrome was observed in ‘nephrocytes’ and was rescued by WT Sply but not by the disease-associated variants. We here discover SGPL1 mutations as a new syndromic form of SRNS. Its pathogenesis entails mesangial cell dysfunction and decrease of active RAC1, which was partially mitigated by S1P receptor inhibition

Mutational analysis of SGPL1: In silico modelling (The analysis results will be shared with Prof. Friedhelm Hildebrandt, Harvard medical school, Division of Nephrology Boston Children's Hospital). These results ( were approved in OAK before), that were shared with Prof. Friedhelm Hildebrandt is now a part of full manuscript for publication with Prof. Friedhelm Hildebrandt as corresponding author . Honnappa Srinivas and Rainer Wilken are the co-authors from NIBR. Please find attached the full manuscript.

Sphingosine-1-phosphate lyase (SGPL1), a key enzyme of sphingolipid metabolism, catalyzes the irreversible decomposition of sphingosine-1-phosphate (S1P) by a retro-aldol fragmentation that yields hexadecanaldehyde and phosphoethanolamine. . Its main substrate sphingosine-1-phosphate (S1P) acts both extracellularly, by binding G protein-coupled receptors of the lysophospholipid receptor family, and inside the cell, as a second messenger. Therefore, S1P takes part in regulating various cellular processes and its levels are tightly regulated. SGPL1 is a symmetric homodimer; two subunits form a tightly intertwined dimer with both chains contributing to the catalytic cavity defined by the covalently bound cofactor pyridoxal phosphate (PLP). Two mutations (R222Q and S346Ile) were found in individuals from families with nephrotic syndrome (unpublished data, Prof. Friedhelm Hildebrandt, Harvard medical school, Division of Nephrology Boston Children's Hospital). To understand the structural changes due to these mutations, we have performed an in silico modelling analysis. The published SGPL1 structure (PDB accession 4Q6R) served as a basis for our in silico model analysis to derive qualitative measure of protein stability and dimer affinity

ReSCoSS: A Flexible Quantum Chemistry Workflow Identifying Relevant Solution Conformers of Drug-like Molecules

Conformational equilibria are at the heart of drug design, yet their energetic description is often hampered by the insufficient accuracy of low-cost methods. Here we present a flexible and semi-automatic workflow based on quantum chemistry, ReSCoSS, designed to identify relevant conformers and predict their equilibria across different solvent environments in the Conductor-like Screening Model for Real Solvents (COSMO-RS) framework. We demonstrate the utility and accuracy of the workflow through conformational case studies on several drug-like molecules from literature where relevant conformations are known. We further show that including ReSCoSS conformers significantly improves COSMO-RS based predictions of physicochemical properties over single-conformation approaches. ReSCoSS has found broad adoption in the in-house drug discovery and development work streams and has contributed to establishing quantum-chemistry methods as a strategic pillar in ligand discovery

Regioselective nitration of 3-fluoro-2-substituted benzoic acids

The preparation of a series of 3-fluoro-2-substituted-6-nitro benzoic acids is described. The partially excellent regioselectivities observed in the nitration step can be rationalized in most cases using Fukui indices of local nucleophilicity

Benchmarking different QM-levels for usage with COSMO-RS

The COSMO-RS method is an established method for the prediction of fluid phase properties such as activity coefficients, liquid-liquid equilibria and free energy of solvation. It is also frequently used in quantum-chemistry based chemical reaction modelling to predict the solvation contribution to the reactions. The COSMOtherm software, which features the currently most advanced implementation of COSMO-RS, is based on quantum-chemical COSMO calculations using the BP86 functional with the def2-TZVPD basis set. As the accuracy of COSMO-RS depends on the accuracy of the underlying quantum-chemical (QC) calculation, it is important to validate the currently used level against other common, presumably superior, approaches such as the more recently developed M06-2x hybrid density functional, or wave-function methods such as MP2. As compared to other applications where the electronic energy is the most important result of the QC calculation, the COSMO-RS method has a much higher dependence on the molecular polarity and thus the electron density distribution. We find that MP2, PBE0 and M06-2X perform slightly worse in fully reparametrized COSMO-RS with respect to the prediction of experimentally measured properties like pKa or logP. Although MP2 was reported to yield better polarities than most DFT functionals for spin unpolarized molecules, this theoretical advantage does not manifest in a practical benefit for the prediction of the properties with the refitted COSMO-RS parameters. Other pure DFT functionals such as PBE or TPSS can be used instead of BP86, but again no practical advantage is expected as they yield extremely similar polarities to the original BP86 calculations

High accuracy quantum-chemistry-based calculation and blind prediction of macroscopic pKa values in the context of the SAMPL6 challenge

Recent advances in the development of low-cost quantum chemical methods have made the prediction of conformational preferences and physicochemical properties of medium-sized drug-like molecules routinely feasible, with significant potential to advance drug discovery. In the context of the SAMPL6 challenge, macroscopic pKa values were blindly predicted for a set of 24 of such molecules. In this paper we present two similar quantum chemical based approaches based on the high accuracy calculation of standard reaction free energies and the subsequent determination of those pKa values via a linear free energy relationship. Both approaches use extensive conformational sampling and apply hybrid and double-hybrid density functional theory with continuum solvation to calculate free energies. The blindly calculated macroscopic pKa values were in excellent agreement with the experiment