Small-Molecule Inhibitors: Disrupting enzyme fluidity
The switching of enzymes between active and inactive states, a process known as enzyme regulation, is crucial in cell biology, and the breakdown of the process has been implicated in many diseases. A number of small-molecule inhibitors work by blocking enzyme function, but efforts to evaluate the efficacy of such inhibitors have been hampered by the lack of a detailed understanding of how they work. For example, some small-molecule inhibitors work by making localized changes to the shape of the enzyme at the site where they bind, whereas others work by inducing changes in another part of the enzyme, a phenomenon known as allostery.
A clearer picture of allostery requires detailed knowledge of enzyme function and the underlying protein dynamics (Henzler-Wildman and Kern, 2007). Now, in eLife, Amy Andreotti (Iowa State University), Thomas Wales (Northeastern University) and colleagues – Raji Joseph, Neha Amatya, Bruce Fulton and John Engen – report on the effects of five different small-molecule inhibitors on an enzyme called Bruton’s tyrosine kinase (BTK; Joseph et al., 2020).
BTK is a kinase that regulates the immune responses of B- and T-cells, and blocking its activity can help suppress inflammatory responses and treat lymphomas and leukemias (Kim, 2019). High-resolution snapshots of the active and inactive states of BTK have been previously obtained using X-ray crystallography (Marcotte et al., 2010; Kuglstatter et al., 2011; Xing and Huang, 2014). Similar to other kinases, an important feature in BTK is a switch called a ‘Glu-Lys switch’ (Taylor et al., 1993). When the critical glutamate (Glu) in the switch is positioned close to a specific lysine (Lys) in the catalytic site, the enzyme is more active. When the enzyme is inactive, it adopts a different shape where the same glutamate is further from the lysine.
While X-ray crystallography can provide information on the active and inactive conformations of enzymes at high resolution, additional techniques are needed to understand how enzymes and small-molecule inhibitors interact in solution, so Joseph et al. combined X-ray crystallography with nuclear magnetic resonance (NMR) and amide hydrogen/deuterium exchange mass spectrometry (HDXMS). NMR provides a global overview of conformation, including the transitions between the active and inactive state, while HDXMS localizes conformational changes at a peptide level.
The experiments revealed that BTK exists in an ensemble of conformations, encompassing the inactive and active states of the enzyme (Figure 1). Joseph et al. then explored how five different small-molecule inhibitors (ibrutinib, dasatinib, GDC-0853, CGI1746 and CC-292) interacted with BTK, and found that each inhibitor resulted in varying ratios of inactive and active conformations. These results establish that BTK changes the likelihood of being in a specific conformation within its ensemble, rather than operating as a discrete on/off switch, which is consistent with the idea that proteins exist in several conformations of varying activity (Onuchic and Wolynes, 2004). This work also supports the view that small-molecule inhibitors may favor certain conformations in an ensemble over others (Boehr et al., 2009; Kar et al., 2010). Joseph et al. also examined a mutation in BTK that confers B-cells with resistance to one of the inhibitors: the cancer drug ibrutinib. They found that this mutation disrupts the inactive conformation of BTK, making the enzyme more active and leading to more aggressive lymphomas that can evade the drug.
This work underscores the limitations of using structural snapshots from X-ray crystallography or cryo-electron microscopy alone to map small-molecule interaction sites, or to describe allosteric effects. It also highlights how combining NMR and HDXMS with static structural data will lead to more complete descriptions of drug-enzyme interactions. More generally, combining structural, dynamic and computational approaches will help researchers to design inhibitor drugs that are not rendered ineffective by disease-resistant mutations.
References
-
The role of dynamic conformational ensembles in biomolecular recognitionNature Chemical Biology 5:789–796.https://doi.org/10.1038/nchembio.232
-
Allostery and population shift in drug discoveryCurrent Opinion in Pharmacology 10:715–722.https://doi.org/10.1016/j.coph.2010.09.002
-
Development of BTK inhibitors for the treatment of B-cell malignanciesArchives of Pharmacal Research 42:171–181.https://doi.org/10.1007/s12272-019-01124-1
-
Theory of protein foldingCurrent Opinion in Structural Biology 14:70–75.https://doi.org/10.1016/j.sbi.2004.01.009
-
cAMP-dependent protein kinase defines a family of enzymesPhilosophical Transactions of the Royal Society of London. Series B, Biological Sciences 340:315–324.https://doi.org/10.1098/rstb.1993.0073
-
Bruton's TK inhibitors: structural insights and evolution of clinical candidatesFuture Medicinal Chemistry 6:675–695.https://doi.org/10.4155/fmc.14.24
Article and author information
Author details
Publication history
Copyright
© 2021, Anand
This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 833
- views
-
- 79
- downloads
-
- 0
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Further reading
-
- Neuroscience
- Structural Biology and Molecular Biophysics
More than 20 recurrent missense gain-of-function (GOF) mutations have been identified in the sodium-activated potassium (KNa) channel gene KCNT1 in patients with severe developmental and epileptic encephalopathies (DEEs), most of which are resistant to current therapies. Defining the neuron types most vulnerable to KCNT1 GOF will advance our understanding of disease mechanisms and provide refined targets for precision therapy efforts. Here, we assessed the effects of heterozygous expression of a Kcnt1 GOF variant (Kcnt1Y777H) on KNa currents and neuronal physiology among cortical glutamatergic and GABAergic neurons in mice, including those expressing vasoactive intestinal polypeptide (VIP), somatostatin (SST), and parvalbumin (PV), to identify and model the pathogenic mechanisms of autosomal dominant KCNT1 GOF variants in DEEs. Although the Kcnt1Y777H variant had no effects on glutamatergic or VIP neuron function, it increased subthreshold KNa currents in both SST and PV neurons but with opposite effects on neuronal output; SST neurons became hypoexcitable with a higher rheobase current and lower action potential (AP) firing frequency, whereas PV neurons became hyperexcitable with a lower rheobase current and higher AP firing frequency. Further neurophysiological and computational modeling experiments showed that the differential effects of the Kcnt1Y777H variant on SST and PV neurons are not likely due to inherent differences in these neuron types, but to an increased persistent sodium current in PV, but not SST, neurons. The Kcnt1Y777H variant also increased excitatory input onto, and chemical and electrical synaptic connectivity between, SST neurons. Together, these data suggest differential pathogenic mechanisms, both direct and compensatory, contribute to disease phenotypes, and provide a salient example of how a pathogenic ion channel variant can cause opposite functional effects in closely related neuron subtypes due to interactions with other ionic conductances.
-
- Structural Biology and Molecular Biophysics
Polycystin-1 (PC1) is the protein product of the PKD1 gene whose mutation causes autosomal dominant Polycystic Kidney Disease (ADPKD). PC1 is an atypical G protein-coupled receptor (GPCR) with an autocatalytic GAIN domain that cleaves PC1 into extracellular N-terminal and membrane-embedded C-terminal (CTF) fragments. Recently, activation of PC1 CTF signaling was shown to be regulated by a stalk tethered agonist (TA), resembling the mechanism observed for adhesion GPCRs. Here, synthetic peptides of the first 9- (p9), 17- (p17), and 21-residues (p21) of the PC1 stalk TA were shown to re-activate signaling by a stalkless CTF mutant in human cell culture assays. Novel Peptide Gaussian accelerated molecular dynamics (Pep-GaMD) simulations elucidated binding conformations of p9, p17, and p21 and revealed multiple specific binding regions to the stalkless CTF. Peptide agonists binding to the TOP domain of PC1 induced close TOP-putative pore loop interactions, a characteristic feature of stalk TA-mediated PC1 CTF activation. Additional sequence coevolution analyses showed the peptide binding regions were consistent with covarying residue pairs identified between the TOP domain and the stalk TA. These insights into the structural dynamic mechanism of PC1 activation by TA peptide agonists provide an in-depth understanding that will facilitate the development of therapeutics targeting PC1 for ADPKD treatment.