Allosteric regulation plays an important role in many biological processes, such as signal transduction, transcriptional regulation, and metabolism. Allostery is rooted in the fundamental physical ...properties of macromolecular systems, but its underlying mechanisms are still poorly understood. A collection of contributions to a recent interdisciplinary CECAM (Center Européen de Calcul Atomique et Moléculaire) workshop is used here to provide an overview of the progress and remaining limitations in the understanding of the mechanistic foundations of allostery gained from computational and experimental analyses of real protein systems and model systems. The main conceptual frameworks instrumental in driving the field are discussed. We illustrate the role of these frameworks in illuminating molecular mechanisms and explaining cellular processes, and describe some of their promising practical applications in engineering molecular sensors and informing drug design efforts.
A collection of contributions to a recent interdisciplinary CECAM (Center Européen de Calcul Atomique et Moléculaire) workshop offers an insightful overview of the understanding of the mechanistic foundations of allostery, gained from computational and experimental analyses of real protein systems and model systems. Various practical applications are illustrated.
In early-stage drug discovery, the hit-to-lead optimization (or “hit expansion”) stage entails starting from a newly identified active compound and improving its potency or other properties. ...Traditionally, this process relies on synthesizing and evaluating a series of analogues to build up structure–activity relationships. Here, we describe a computational strategy focused on kinase inhibitors, intended to expedite the process of identifying analogues with improved potency. Our protocol begins from an inhibitor of the target kinase and generalizes the synthetic route used to access it. By searching for commercially available replacements for the individual building blocks used to make the parent inhibitor, we compile an enumerated library of compounds that can be accessed using the same chemical transformations; these huge libraries can exceed many millionsor billionsof compounds. Because the resulting libraries are much too large for explicit virtual screening, we instead consider alternate approaches to identify the top-scoring compounds. We find that contributions from individual substituents are well described by a pairwise additivity approximation, provided that the corresponding fragments position their shared core in precisely the same way relative to the binding site. This key insight allows us to determine which fragments are suitable for merging into single new compounds and which are not. Further, the use of pairwise approximation allows interaction energies to be assigned to each compound in the library without the need for any further structure-based modeling: interaction energies instead can be reliably estimated from the energies of the component fragments, and the reduced computational requirements allow for flexible energy minimizations that allow the kinase to respond to each substitution. We demonstrate this protocol using libraries built from six representative kinase inhibitors drawn from the literature, which target five different kinases: CDK9, CHK1, CDK2, EGFRT790M, and ACK1. In each example, the enumerated library includes additional analogues reported by the original study to have activity, and these analogues are successfully prioritized within the library. We envision that the insights from this work can facilitate the rapid assembly and screening of increasingly large libraries for focused hit-to-lead optimization. To enable adoption of these methods and to encourage further analyses, we disseminate the computational tools needed to deploy this protocol.
Small-molecules that inhibit interactions between specific pairs of proteins have long represented a promising avenue for therapeutic intervention in a variety of settings. Structural studies have ...shown that in many cases, the inhibitor-bound protein adopts a conformation that is distinct from its unbound and its protein-bound conformations. This plasticity of the protein surface presents a major challenge in predicting which members of a protein family will be inhibited by a given ligand. Here, we use biased simulations of Bcl-2-family proteins to generate ensembles of low-energy conformations that contain surface pockets suitable for small molecule binding. We find that the resulting conformational ensembles include surface pockets that mimic those observed in inhibitor-bound crystal structures. Next, we find that the ensembles generated using different members of this protein family are overlapping but distinct, and that the activity of a given compound against a particular family member (ligand selectivity) can be predicted from whether the corresponding ensemble samples a complementary surface pocket. Finally, we find that each ensemble includes certain surface pockets that are not shared by any other family member: while no inhibitors have yet been identified to take advantage of these pockets, we expect that chemical scaffolds complementing these "distinct" pockets will prove highly selective for their targets. The opportunity to achieve target selectivity within a protein family by exploiting differences in surface fluctuations represents a new paradigm that may facilitate design of family-selective small-molecule inhibitors of protein-protein interactions.
Conventionally, most amino acid substitutions at “important” protein positions are expected to abolish function. However, in several soluble-globular proteins, we identified a class of nonconserved ...positions for which various substitutions produced progressive functional changes; we consider these evolutionary “rheostats”. Here, we report a strong rheostat position in the integral membrane protein, Na+/taurocholate (TCA) cotransporting polypeptide, at the site of a pharmacologically relevant polymorphism (S267F). Functional studies were performed for all 20 substitutions (S267X) with three substrates (TCA, estrone-3-sulfate, and rosuvastatin). The S267X set showed strong rheostatic effects on overall transport, and individual substitutions showed varied effects on transport kinetics (Km and Vmax) and substrate specificity. To assess protein stability, we measured surface expression and used the Rosetta software (https://www.rosettacommons.org) suite to model structure and stability changes of S267X. Although buried near the substrate-binding site, S267X substitutions were easily accommodated in the Na+/TCA cotransporting polypeptide structure model. Across the modest range of changes, calculated stabilities correlated with surface-expression differences, but neither parameter correlated with altered transport. Thus, substitutions at rheostat position 267 had wide-ranging effects on the phenotype of this integral membrane protein. We further propose that polymorphic positions in other proteins might be locations of rheostat positions.
Cyclin-dependent kinase 9 (CDK9) promotes transcriptional elongation through RNAPII pause release. We now report that CDK9 is also essential for maintaining gene silencing at heterochromatic loci. ...Through a live cell drug screen with genetic confirmation, we discovered that CDK9 inhibition reactivates epigenetically silenced genes in cancer, leading to restored tumor suppressor gene expression, cell differentiation, and activation of endogenous retrovirus genes. CDK9 inhibition dephosphorylates the SWI/SNF protein BRG1, which contributes to gene reactivation. By optimization through gene expression, we developed a highly selective CDK9 inhibitor (MC180295, IC50 = 5 nM) that has broad anti-cancer activity in vitro and is effective in in vivo cancer models. Additionally, CDK9 inhibition sensitizes to the immune checkpoint inhibitor α-PD-1 in vivo, making it an excellent target for epigenetic therapy of cancer.
Display omitted
•CDK9 inhibition reactivates epigenetically silenced genes in multiple cancer cells•CDK9 binds to and phosphorylates BRG1, which contributes to gene silencing•We developed a novel potent and selective CDK9 inhibitor, MC180295•MC180295 shows promising anti-tumoral and immunosensitization activity
Inhibition of a kinase typically associated with transcription elongation complexes leads to reactivation of tumor suppressor genes and increases sensitivity to immunotherapy in cancer models.
Abstract
The p53 tumor suppressor gene plays a central role in human cancer. In addition to mutation, there are genetic coding region variants of p53 that exist in different ethnic groups. In some ...cases, these variants have incomplete or impaired function and confer increased cancer risk. We call such variants ‘hypomorphs’. Two p53 hypomorphs exist in populations of African descent: P47S and Y107H. Our research on the P47S hypomorph of p53 revealed that this variant confers increased risk for cancer in humans and in mouse models (Jennis, Genes Dev 2016; Murphy, NPJ Breast Cancer 2017). We find that P47S protein is selectively impaired for the ability to confer sensitivity to ferroptosis (Leu, PNAS 2019); consequently, we find that human and mouse cells with this variant accumulate iron in certain tissues, and that the P47S variant is associated with iron overload in African Americans (Singh, Nat Comm 2020). We find that tumor cells containing the P47S variant are resistant to most genotoxic chemotherapies, but we have discovered that P47S tumor cells are markedly more sensitive to other therapies, including cisplatin and BRD2/4 inhibitors (Barnoud, Can Res 2018). Our current focus is on another African-centric p53 hypomorph, Y107H. We show that this variant is impaired for p53-mediated apoptosis, and is associated with cancer risk in a mouse model. We also show that Y107H is impaired for the transactivation of only a dozen p53 target genes. Notably, half of these genes are known tumor suppressor genes. Our biophysical and NMR structure of the Y107H protein explains the mechanistic basis for the impaired function of this hypomorph. Our analysis of the P47S and Y107H hypomorphs of p53 has significant potential to understand cancer risk and improve cancer therapy in populations of African descent.
Citation Format: Tim Barnoud, Jessica Leung, Keerthana Gnanapradeepan, Lei Ke, John Karanicolas, Maureen E. Murphy. African-centric p53 hypomorphs play significant roles in cancer risk and efficacy of therapy abstract. In: Proceedings of the AACR Virtual Conference: Thirteenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2020 Oct 2-4. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(12 Suppl):Abstract nr PR08.
We have recently completed a full re-architecturing of the ROSETTA molecular modeling program, generalizing and expanding its existing functionality. The new architecture enables the rapid ...prototyping of novel protocols by providing easy-to-use interfaces to powerful tools for molecular modeling. The source code of this rearchitecturing has been released as ROSETTA3 and is freely available for academic use. At the time of its release, it contained 470,000 lines of code. Counting currently unpublished protocols at the time of this writing, the source includes 1,285,000 lines. Its rapid growth is a testament to its ease of use. This chapter describes the requirements for our new architecture, justifies the design decisions, sketches out central classes, and highlights a few of the common tasks that the new software can perform.
Abstract only
Background
Human Na
+
/taurocholate cotransporting polypeptide (NTCP) is a sodium dependent bile acid transporter expressed at the basolateral membrane of human hepatocytes. It is ...important for the enterohepatic circulation of bile acids, such as taurocholate (TCA), but it also transports other compounds including estrone‐3‐sulfate (E3S) and rosuvastatin. We previously identified a rheostat position at a naturally occurring polymorphism, S267F. That is, when other amino acids were substituted into this position, progressive changes in function were observed.
Aim
The purpose of the current study is to determine whether position N271, a predicted rheostat, is indeed another rheostat position in NTCP.
Methods
The crystal structures of two bacterial NTCP homologs were used to model the structure of human NTCP in the inward‐open and outward‐open conformations. Mutations were then introduced into the structural models and the energetic effect on protein stability was evaluated using the Rosetta software. Based on these calculations we predicted that N271 is a rheostat. Mutants for position N271 were generated using site‐directed mutagenesis and the resulting proteins were transiently expressed in HEK293 cells. The functions of the mutants were characterized by measuring uptake of radiolabeled model substrates:
3
H‐taurocholate,
3
H‐estrone‐3‐sulfate, and
3
H‐rosuvastatin. Plasma membrane expression levels were determined by surface biotinylation assays.
Results
Initial transport studies using nanomolar concentrations of TCA, E3S, and rosuvastatin indicated a variety of intermediate functional outcomes for the mutants. Furthermore, this alteration in function was substrate dependent, which is consistent with our findings for S267. Some mutants, such as N271A, transport substrates similar to wild‐type NTCP, while others, like N271K, show variation depending on the substrate. N271P consistently transports all substrates minimally. In addition, there is no correlation between the initial uptake results and the Rosetta energies.
Summary and Conclusions
Initial transport studies demonstrate that position N271 is a rheostat position, as predicted by the alteration in Rosetta energy scores. However, there was no correlation between the calculated energy scores and the functional results. This suggests that functional outcomes of specific amino acid replacements at position N271 may arise from more complex conformational changes than were captured by the two modeled states. Additional experiments should help to determine to what extent the observed change in function is due to variations in the substrate affinity, the turnover number or the transport capacity of the mutants.
Support or Funding Information
P30 GM118247, GM077336, and W. M. Keck Foundation
Natural products have served as an inspiration to scientists both for their complex three-dimensional architecture and exquisite biological activity. Promysalin is one such Pseudomonad secondary ...metabolite that exhibits narrow-spectrum antibacterial activity, originally isolated from the rhizosphere. We herein utilize affinity-based protein profiling (AfBPP) to identify succinate dehydrogenase (Sdh) as the biological target of the natural product. The target was further validated in silico, in vitro, in vivo, and through the selection, and sequencing, of a resistant mutant. Succinate dehydrogenase plays an essential role in primary metabolism of Pseudomonas aeruginosa as the only enzyme that is involved both in the tricarboxylic acid cycle (TCA) and in respiration via the electron transport chain. These findings add credence to other studies that suggest that the TCA cycle is an understudied target in the development of novel therapeutics to combat P. aeruginosa, a significant pathogen in clinical settings.
PDF
