Application of In Silico Filtering and Isothermal Titration Calorimetry for the Discovery of Small Molecule Inhibitors of MDM2

The initial discovery phase of protein modulators, which consists of filtering molecular libraries and in vitro direct binding validation, is central in drug discovery. Thus, virtual screening of large molecular libraries, together with the evaluation of binding affinity by isothermal calorimetry, generates an efficient experimental setup. Herein, we applied virtual screening for discovering small molecule inhibitors of MDM2, a major negative regulator of the tumor suppressor p53, and thus a promising therapeutic target. A library of 20 million small molecules was screened against an averaged model derived from multiple structural conformations of MDM2 based on published structures. Selected molecules originating from the computational filtering were tested in vitro for their direct binding to MDM2 via isothermal titration calorimetry. Three new molecules, representing distinct chemical scaffolds, showed binding to MDM2. These were further evaluated by exploring structure-similar chemical analogues. Two scaffolds were further evaluated by de novo synthesis of molecules derived from the initial molecules that bound MDM2, one with a central oxoazetidine acetamide and one with benzene sulfonamide. Several molecules derived from these scaffolds increased wild-type p53 activity in MCF7 cancer cells. These set a basis for further chemical optimization and the development of new chemical entities as anticancer drugs

Collagenase Administration into Periodontal Ligament Reduces the Forces Required for Tooth Extraction in an Ex situ Porcine Jaw Model

Minimally invasive exodontia is among the long-sought-for development aims of safe dental medicine. In this paper, we aim, for the first time, to examine whether the enzymatic disruption of the periodontal ligament fibers reduces the force required for tooth extraction. To this end, recombinantly expressed clostridial collagenase G variant purified from Escherichia coli was injected into the periodontal ligament of mesial and distal roots of the first and second split porcine mandibular premolars. The vehicle solution was injected into the corresponding roots on the contralateral side. Following sixteen hours, the treated mandibles were mounted on a loading machine to measure the extraction force. In addition, the effect of the enzyme on the viability of different cell types was evaluated. An average reduction of 20% in the applied force (albeit with a large variability of 50 to 370 newton) was observed for the enzymatically treated roots, reaching up to 50% reduction in some cases. Importantly, the enzyme showed only a minor and transient effect on cellular viability, without any signs of toxicity. Using an innovative model enabling the analytical measurement of extraction forces, we show, for the first time, that the enzymatic disruption of periodontal ligament fibers substantially reduces the force required for tooth extraction. This novel technique brings us closer to atraumatic exodontia, potentially reducing intra- and post-operative complications and facilitating subsequent implant placement. The development of novel enzymes with enhanced activity may further simplify the tooth extraction process and present additional clinical relevance for the broad range of implications in the oral cavity

Structural Dynamics of the Potassium Channel Blocker ShK: SRLS Analysis of ¹⁵N Relaxation

The 35-residue ShK peptide binds with high affinity to voltage-gated potassium channels. The dynamics of the binding surface was studied recently with (microsecond to millisecond) ¹⁵N relaxation dispersion and (picosecond to nanosecond) ¹⁵N spin relaxation of the N–H bonds. Relaxation dispersion revealed microsecond conformational-exchange-mediated exposure of the functionally important Y23 side chain to the peptide surface. The spin relaxation parameters acquired at 14.1 and 16.45 T have been subjected to model-free (MF) analysis, which yielded a squared generalized order parameter, <i>S</i>², of approximately 0.85 for virtually all of the N–H bonds. Only a “rigid backbone” evaluation could be inferred. We ascribe this limited information to the simplicity of MF in the context of challenging data. To improve the analysis, we apply the slowly relaxing local structure (SRLS) approach, which is a generalization of MF. SRLS describes N–H bond dynamics in ShK in terms of a local potential, <i>u</i>, ranging from 10 to 18.5 <i>k</i>_B<i>T</i>, and a local diffusion rate, <i>D</i>₂, ranging from 4.2 × 10⁸ to 2.4 × 10¹⁰ s^–1. This analysis shows that <i>u</i> is outstandingly strong for Y23 and relatively weak for K22, whereas <i>D</i>₂ is slow for Y23 and fast for K22. These observations are relevant functionally because of the key role of the K22–Y23 dyad in ShK binding to potassium channels. The disulfide-bond network exhibits a medium-strength potential and an alternating wave-like <i>D</i>₂ pattern. This is indicative of moderate structural restraints and motional plasticity, in support of, although not directly correlated with, the microsecond binding-related conformational exchange process detected previously. Thus, new information on functionally important residues in ShK and its overall conformational stability emerged from the SRLS analysis, as compared with the previous MF-based estimate of backbone dynamics as backbone rigidity