Multiple myeloma (MM) is a cytogenetically heterogeneous clonal plasma cell proliferative disorder that accounts for approximately 10%-15% of all hematological malignancies [1,2]. In most countries, MM is the second most common hematological malignancy and has exhibited a steadily increasing incidence rate over the past three or four decades globally [2]. MM treatment has recently made remarkable progress. With many new agents, such as novel selective inhibitors of nuclear export (the XPO1 inhibitor selinexor), monoclonal antibodies and chimeric antigen receptor T (CAR-T) cell therapy, being applied in MM treatment, overall survival has been significantly improved [3,4]. However, MM remains incurable today, and most patients relapse and become refractory to current treatments, prompting a continuous search for novel therapeutics [5].

Homo sapiens caseinolytic protease P (HsClpP), a mitochondrial serine protease, plays an essential role in maintaining mitochondrial homeostasis and the integrity of oxidative phosphorylation by regulating mitochondrial protein folding and degradation [[6], [7], [8]]. HsClpP is over-expressed in various solid tumors and hematological malignancies, and is implicated in sustaining tumor cell viability, driving proliferation, conferring chemoresistance, and promoting metastasis [[9], [10], [11]]. As reported, pharmacological hyperactivation of HsClpP can disrupt oxidative phosphorylation and exert anticancer effects, positioning HsClpP as a compelling therapeutic target in various tumors [[12], [13], [14]]. However, the role and translational potential of HsClpP activation in MM treatment remain poorly characterized. Recently, the expression of HsClpP was found to be higher in MM patients than in healthy participants, and high expression of HsClpP was associated with poor prognosis in MM [15,16]. Moreover, some previous studies demonstrated that mitochondrial biogenesis gene expression increased and metabolism shifted toward oxidative phosphorylation in some relapsed and refractory MM (RRMM) [13,17,18]. These results imply the therapeutic potential of HsClpP activation in MM.

Previously, a series of classical small-molecule HsClpP activators has been identified, including ADEP-41 [19], D9 [20], TR-57 [21,22], ONC201 [23], 16z [24], ZG111 [25,26], ZK53 [27], NCA029 [28], 7K [29], SL44 [30], CLPP-1071 [31] and CLPP-2068 [32] (Fig. 1). Among them, ONC201 stands out for its remarkable antiproliferative and pro-apoptotic effects across various tumors [33,34]. Using ONC201 as the lead compound, a series of structural modifications have been reported, such as 7k [29] (a ring-opened analog of ONC201) and XT6 [35] (a derivative featuring the introduction of a double bond into the five-membered ring scaffold of ONC201, Fig. 1), both of which exhibit enhanced antitumor efficacy.

Recently, we found that D215, an HsClpP activator reported in our previous study (named 7e) [29], showed cytotoxic effects against MM cells (AMO.1) (Fig. 2A). Its HsClpP agonistic potency was further validated by triggering the degradation of both short peptide (AC-WLA-AMC) and protein substrates (α-casein) (Fig. 2B, C, D). However, compound D215 exhibited a sub-optimal safety profile, as evidenced by its concerning inhibitory activity against human normal cells. Thus, chemical structural optimization would be an ideal strategy to enhance its bio-activity and minimize toxicity. In our work, structural modification based on D215 yielded a series of derivatives, among which GU18 had remarkable anti-MM efficacy with nanomolar-range IC50 values against MM cells, representing a significant enhancement compared to the micromolar activity of D215. Furthermore, GU18 demonstrated selective cytotoxicity against MM cells over normal cells, favorable pharmacokinetics, and was well-tolerated in vivo. Most importantly, in MM xenograft models, GU18 achieved a remarkable 91.4% tumor growth inhibition at a dosage of 30 mg/kg every other day. Generally, our study not only provides a drug candidate for the treatment of MM but also suggests HsClpP as a potential therapeutic target for MM.