Enterovirus 71 (EV71) belongs to the Picornavirus family of viruses. It causes neurological diseases in adults and the hand, foot, and mouth disease in children. Outbreaks of EV71 have emerged as a global health issue due to severe neurological complications leading to fatal encephalitis in a relevant number of cases. The viral genome, constituted by a single-stranded RNA molecule, is translated into a large polyprotein inside the host cell. The virally encoded protease 3Cpro plays a crucial role in the proliferation of this virus and represents a promising drug target for the design of antiviral drugs ^[1][2][19,20].

Wang et al. investigated different warheads in order to improve the stability and selectivity of a previously developed set of inhibitors of the 3Cpro enzyme of EV71 that were originally designed with an aldehyde warhead ^[3][4][21,22]. They introduced a panel of warheads, such as nitrile, ketoamide, alcohol, hydroxyl amide, doubly activated olefins, and hydroxy-nitrile groups, as an alternative to the aldehyde group. In this case, the nitrile warhead led to a moderately active inhibitor (4), while an α-hydroxynitrile gave better selectivity toward cysteine vs. serine protease and cell-based activity (inhibitor 5, Figure 12). An X-ray crystal structure of the enzyme-bound inhibitor revealed a non-covalent binding mode ^[4][22].

A different example of a warhead was introduced in compound 6. The electron-withdrawing properties of the cyano group were exploited for the preparation of double-activated olefins that are able to function as Michael acceptors upon reaction with the cysteine active site residue of EV71 3Cpro. The cyano group, which, in this case, does not form a covalent bond with the active site cysteine but contributes to increasing the reactivity of the olefin carbon, provided more selective enzyme inhibitory activity coupled to a better and broad-spectrum activity on cell-based assays (6) ^[5][6][23,24].

MPro is a cysteine protease with an important role in the SARS-CoV-2 replicative cycle, and since the SARS-CoV-2 epidemic outbreak in 2019, intense research efforts have been devoted by scientists in the search for MPro inhibitors ^[7][8][9][25,26,27]. These efforts culminated with the discovery of the drug Paxlovid, which is a combination of the Mpro inhibitor nirmatrelvir  (3, Figure 1) with the metabolic booster ritonavir. Nirmatrelvir (3) was developed based on a structure-based optimization approach aimed at optimizing the interaction with the S1 and S2 pockets and, at the same time, monitoring the solubility and selected pharmacokinetic properties of the designated hits. The selection of 3 to be advanced in preclinical optimization over the benzothiazole analogue 7 was mainly supported by improved water solubility and a lower propensity to epimerization at the α-carbon, attributable to the presence of the cyano group instead of the benzothiazole moiety ^[10][15].

The X-ray co-crystal structures of SARS-CoV-2 Mpro in complex with nirmatrelvir confirmed the formation of a reversible covalent Cys145 adduct with the nitrile substituent ^[10][11][15,16]. It is worth mentioning that this inhibitor has a similar efficacy toward the recently emerged SARS-CoV-2 variants ^[12][28].

Briefly, the crystal structure of Mpro in complex with nirmatrelvir (3) showed a full occupancy of the ligand within the active site of the enzyme, targeting the S1-S4 subsites, as illustrated in Figure 23. The nitrile functional group interacts at the S1′ subsite, forming a covalent adduct with the reactive Cys145 of the enzyme. In addition, H bonds with the backbone of Cys145 and His164 are evident. The pyrrolidinone moiety strongly targets the S1 subsite, establishing a series of H bonds with the backbone of Phe140 and the sidechains of Glu166 and His163. The azabicyclo moiety interacts at the S2 subsite by hydrophobic contacts, while the N-terminal moiety of the molecule can establish a series of H bonds with the backbone of Glu166. The portion bearing the trifluoro function targets residues at the S3 and S4 subsites, forming hydrophobic contacts.

In the study by Bai et al. ^[13][29] a series of inhibitors typified by 8 (Figure 12) are described and, also in this study, the nitrile warhead was pointed out as a good alternative to the aldehyde warhead to achieve activity against the Mpro enzyme. Importantly, this study reports the selectivity of the compounds bearing a nitrile warhead vs. human cathepsins B, S, and L, suggesting that the designed compounds bearing a nitrile warhead were significantly less active in inhibiting cathepsins S and L than the hydroxymethylketon warhead, even though further studies are needed to support this observation.

A different warhead has been described by Bridenbach and colleagues ^[14][30]. In their search for potent SARS-CoV-2 Mpro inhibitors, two classes of compounds were reported, one of them being based on an azanitrile warhead (9). The authors also confirmed that these azanitrile inhibitors were able to covalently interact with the Mpro catalytic cysteine moiety.

Cruzain (Cz) is the major cysteine protease of T. cruzi ^[15][31], the causative agent of Chagas disease, whose therapy is mainly based on the use of two drugs: benznidazole and nifurtimox. Both these drugs are responsible for serious side effects and are ineffective against the chronic stage of the disease ^[16][32]. Innovative therapeutic approaches are urgently needed for the treatment of this disease that affects one million people worldwide, with major diffusion in Latin American ^[17][33]. Cz has been explored for several years as a drug target, and nitrile-based inhibitors have been deeply investigated ^[15][31]. The most recent examples are reported below.

Human African trypanosomiasis (HAT), also defined as sleeping sickness, is a parasitic disease that mainly affects sub-Saharan Africa. The parasite responsible for the disease is Trypanosoma brucei, for which two subspecies are known: T. b. gambiense and T. b. rhodesiense, leading to different symptomatology. The cysteine protease of T. brucei, namely rhodesain (RD), has been explored as a drug target through the design and synthesis of nitrile-based peptidomimetics ^[18][19][8,45].

Leishmaniasis is a vector-borne parasitic disease with a widespread distribution in tropical and subtropical countries, including the Mediterranean region. It has been reported that from 700,000 to 1 million new cases of leishmaniasis occur annually, while the chemotherapy for this infection is largely inadequate ^[20][21][49,50]. An attractive biological target for therapeutic intervention in the treatment of leishmaniasis has been identified in the cysteine protease B (CPB) expressed by this pathogenic protozoan.

Toxoplasma gondii is a neurotrophic protozoan parasite that exists in two principal forms, the tachyzoite and bradyzoite, this latter form being responsible for the chronic infection during which cysts form within the central nervous system (CNS) and muscle tissue to establish a life-long infection. T. gondii cathepsin protease L (TgCPL) is critical to parasite survival during the chronic phase of infection ^[22][23][52,53]. So, the development of effective and brain-penetrant inhibitors could lead to a great advance in the chemotherapy of this parasitic infection, which is currently ineffective against the chronic form.