Heat shock proteins (HSPs) play a central role in the clearance of damaged proteins by inducing protein aggregation and proteotoxicity. This process occurs by preventing inappropriate stress-induced protein aggregation, ensure proper refolding of denatured proteins, and, if necessary, the promotion of their degradation [1
]. Recent studies have proven that increased protein synthesis (translation) is vital to the conversion of neoplasms. As a result of this increase, cancer cells appear to be particularly susceptible to agents that inhibit the removal of aggregated or misfolded proteins generated by protein synthesis as a product [4
]. Hsp70 protein member families are among the highly conserved proteins and play a critical role in these processes [7
]. The primary stress-inducing member of the Hsp70 chaperone family is known as Hsp72 and is encoded by two genes, HSPA1A and HSPA1B, which generate isoforms of Hsp72 [8
]. Hsp72 is extremely homologous to the 78 kDa glucose-regulated protein, which plays a significant role in organizing the unfolding protein response [9
]. Hsp72 is expressed at high levels in malignant tumors of various origins [10
] and enhances cancer cell survival [11
]. Thus, the inhibition of Hsp72 is considered to be a successful pathway in anti-tumor therapy [13
All the different functions of Hsp70s are accomplished through a transient chaperone interaction with substrate proteins through its C-terminal substrate-binding domain (SBD) [14
]. The nucleotide binds allosterically to the N
-terminal nucleotide-binding domain (NBD) to control the transient chaperone interaction. The affinity of the SBD for substrates decreases by 10- to 400-fold when ATP is binding to the NBD. Hence, the inhibition of NBD is considered one of the most promising strategies for HSP72 function inhibition [15
]. The NBD consists of two adjacent lobes (lobe I and lobe II), which form a deep nucleotide groove connected to the base. Each lobe consists of two subdomains (IA, IIA, IB, and IIB) [16
]. Domains IB and IIB are linked to IA and IIA, respectively, by flexible hinges and control access to the nucleotide-binding sites [18
] (Figure 1
Several studies have designed potential Hsp72 inhibitors, including 2-phenylethynesulfonamide (PES) [19
], 15-deoxyspergualin (DSG) [20
], natural products Oridonin [21
] and Novolactone [22
], but upregulation is one of the most challenges associated with drug resistance and poor clinical outcomes [23
]. The challenging hurdle to cellular activity for competitive nucleotide inhibitors of HSP72 is due to the highly conserved domain. This conserved domain is mostly occupied by ADP and ATP (ADP, KD ~ 110 nm) in addition to hydrophilic and electrostatic interactions with the nucleotide ribose and phosphate amino acid residues, hence difficult drug binders [24
]. Covalent inhibition is a key approach for high-affinity proteins [25
] and has recently sparked interest among the community of pharmaceutical research [26
]. Covalent inhibition occurs when the electrophilic moiety of a covalent ligand connects with a nucleophilic residue of a biological target, resulting in an irreversible link between the protein and the drug [8
]. For example, it can inhibit the same biological target at a lower concentration than a noncovalent drug due to the long-lasting effects of a covalent drug [27
]. An example of a covalent reaction between a ligand and its protein target is shown in Figure 2
In a recent study by Pettinger et al. (2017) using fluorescence polarization (FP) assay and crystallography, the authors observed an unexpected covalent bond interaction between 8-N-benzyladenosine and lysine-56 of the NBD of HSP72 (HSP72-NBD domain). This unexpected covalent bond interaction resulted in the arrest of the NBD via hydrogen-bonding array of the ribose and adenine moieties with the lipophilic para-chlorobenzylamine moiety, parallel with the two α-helices of the binding cavity [29
] (Figure 3
). It is worth highlighting that the observed covalent inhibition of HSP72 via lysine-56 by 8-N
-benzyladenosine was opposed to the anticipated 8-N
-benzyladenosine covalent inhibition of HSP72 via cysteine-17.
The unexpected preferential covalent bond formation of 8-N-benzyladenosine with lysine-56 over cysteine-17 prompted the need to investigate the conformational plasticity and structural dynamics associated with this unexpected covalent interaction. To accomplish this, we utilized in silico approaches such as covalent molecular dynamics simulation, clustering and principal component analyses to define and compare the structural dynamics of 8-N-benzyladenosine-Lys-56 modeled covalent complex with 8-N-benzyladenosine-cys-17 covalent complex on HSP72-NBD domains.
Extensive analyses reveal that the coupling of the inhibitor to cysteine-17 is intrinsically more dynamic than to lysine-56, mainly in the IIA and IA α-helices region. Conformational dynamics analysis further reveals that the coupling of 8-N-benzyladenosine to lysine-56 induces a closed conformation of the IIB and IIA α-helices of the NBD. In contrast, an open conformation was observed when coupled to the cysteine-17 residue.
The human heat shock protein 72 (HSP72) represents a vital therapeutic target during the critical stages of oncogenesis and progression of human cancers. In this study, we performed covalent molecular dynamic simulation followed by extensive analyses to decipher the structural basis and conformational dynamics associated with the unexpected preferential coupling of 8-N-benzyladenosine to lysine-56 over cysteine-17 in HSP72-NBD models. The results reveal that the irreversible binding of 8-N-benzyladenosine to lysine-56 over cysteine-17 in HSP72-NBD represents the most stable conformation with minimal intrinsic dynamics. Clustering and PCA showed that the first three principal components accounted for 67.8% and 53.2% of the total variance for Cys-17-HSP72 and Lys-56-HSP72 covalent complexes, respectively. The magnitude of principal component 1 (PC1) was observed to be the highest for the Cys-17-HSP72 complex (51.2%); however, a relatively lower PC1 of 30.8% was observed for Lys-56-HSP72 covalent complex.
The conformational dynamics analysis further reveals what the experimental study could not capture and explain, that the coupling of 8-N-benzyladenosine to Lysine-56 induces a closed conformation of the IIB and IIA α-helices of the nucleotide-binding subdomain. In contrast, an open conformation was observed in coupling to Cysteine-17 residue. Interestingly, the close conformation maintained the crucial salt-bridge between Glu-268 and Lys-56 residues, which strengthens the interaction affinity of 8-N-benzyladenosine nearly identical to ADP/Pi bound to the HSP72-NBD. It is rare for non-catalytic lysine residues to form a covalent bond with an inhibitor. The recent unexpected covalent formation between lysine-56 and 8-N-benzyladenosiness would assist with the design of more potent and highly selective covalent inhibitors for HSP72 with the potential to overcome drug resistance challenges and represent a novel therapeutic approach for inhibiting HSP72 oncoprotein.