The search for new antibiotics against drug-resistant microbes has led to renewed focus on antimicrobial peptides (AMPs) and synthetic small peptides as anti-infective agents [[13], [14], [15], [16], [17], [18]]. Antimicrobial peptides (AMPs) are known to exhibit broad antimicrobial activity and have received attention because many bacteria, including mycobacterial, are less prone to developing resistance against them, due in part to the non-specific mode of action of AMPs. Peptide and peptide-derived drugs account for a significant portion of today's pharmaceuticals market. In fact, from 2013 to 2019, sell of peptide drugs have grown two-fold [13,19]. Unsurprisingly, the most success has been in development and use of peptide drugs that mimic the action of glucagon-like peptide-1 (GLP-1) hormone, which stimulates insulin secretion, lowers blood glucose levels, and are effective in treatment of type 2 diabetes mellitus (T2DM) [19,20].

According to the antimicrobial peptide database (APD, http://aps.unmc.edu/AP/), there are over 3940 peptides as at January 2024, which includes 3146 natural antimicrobial peptides (AMPs), 190 predicted and 314 synthetic AMPs with various biological activities [21]. These different AMPs are marked by differences in physicochemical properties such as net charge, structure, and solubility. However, most AMPs are amphipathic and cationic. While the positive charge allows AMPs to interact successfully with microbial membranes, their amphipathic properties allow them to insert into the bacterial membrane, which leads to pore formation resulting in cell death via osmotic shock. Additionally, some AMPs are also able to pass through cellular membrane to exhibit intracellular activities by blocking cell wall synthesis, protein synthesis, nucleic acid synthesis, and enzymatic activities. Therefore, on the basis of their antimicrobial mechanism, AMPs are generally classified into membrane acting and non-membrane acting peptides [13,17,20,22]. Membrane acting AMPs disrupt the membrane, while peptides with non-membrane permeabilizing modes of action translocate across the membrane without causing damage. A few examples of membrane acting AMPs capable of creating trans-membrane pores on the target membrane include defensin, melittin, magainins, and LL-37 [[23], [24], [25]]. Non-membrane AMPs which are known to translocate across the cell membrane and disrupt normal cell functioning include buforin II, dermaseptin, HNP-1, pleurocidin, indolicidin, pyrrhocoricin, and mersacidin [20,26]. Put together, the broad-spectrum activity of AMPs against a wide range of microorganisms and their low propensity towards microbial resistance make them very attractive candidates for development of new antimicrobial agents, especially in combating drug resistant mycobacteria.

However, AMPs have important drawbacks which have limited their use in development of new antimicrobial agents, including development of new TB drugs. They are readily hydrolyzed by proteases in vivo, which lowers their bioavailability. Furthermore, the high cost associated with their synthesis has limited their production and adoption as therapeutic agents by pharmaceutical companies. Additionally, AMPs are unstable in certain pHs and demonstrate possible immunogenicity and toxicity [17]. Therefore, there is a growing interest in the design and development of non-natural mimics of AMPs with better bioavailability and biostability.

Oligo-N-substituted glycines (peptoids) are now seen as favorable alternatives to AMPs [[27], [28], [29]]. Whereas peptoids have similar backbone atom sequences as AMPs, they are less vulnerable to enzymatic and protease degradation. Unlike AMPs which have side chains attached to the alpha carbon, peptoid functional side chains are anchored to the nitrogen (N)-atom (Fig. 1). Since there is no investigated protease which is known to recognize and degrade them, this makes peptoids more stable and more suitable for pharmaceutical development [28,30,31]. Another distinct advantage of peptoids over AMPs is the fact there are more opportunities for different kinds of primary amines to be incorporated as side chains into a peptoid chain leading to a much larger library of new peptoid analogs, which is not possible by merely modifying conventional AMPs. In addition, peptoids have been shown to exhibit low cytotoxicity relative to AMPs, further strengthening their potential therapeutic application [17,28,30].

While there are a growing number of α-peptoids synthesized and studied for their antimicrobial activity, there are relatively very few β-peptoids studied. Yet, it has long been known that although β-amino acids naturally occurring peptides are rare, their extra methylene group makes them unable to be recognized easily by traditional proteases, rendering them intrinsically more stable [32,33]. Additionally, studies have also shown that peptido mimics composed of alternating α- and β-amino acid residues not only exhibited significant antimicrobial activity, but are also very resistant towards peptidase degradation by trypsin, pronase, and chymotrypsin [32,34]. Although a few studies have focused on the synthesis and properties of peptoid-based ligands, the effect of their combined α- and β-peptoid residues on the secondary structures and folding propensity of peptoids [35,36], and their general antimicrobial activity [32,36,37], there are relatively very few or no studies evaluating the antimycobacterial activity of β-peptoids [17,38]. Therefore, this study aims to bridge this gap by synthesizing and evaluating several α- and β-petoids as antitubercular agents.

Therefore, in this study, we report the design, synthesis and biological evaluation of new peptoids against drug-susceptible and drug-resistant strains of M. tb in our search for selective inhibitors of MDR-M. tb. The design and choice of new peptoids were informed by the established fact that peptoids act similar to AMPs by either disrupting the mycobacterial cell wall or translocating the cell wall in order to inhibit important biological pathways critical to bacterial survival [17,22,28].

In fact, the thick, multi-layered, extremely hydrophobic cell envelope, which results in very low cellular permeability of antibiotic agents, acts as a barrier against many classes of hydrophilic antibacterial drugs [1,4,39]. Consequently, the mycobacterial cell wall is very important for the long-term survival of M. tb in the hostile environment of the macrophage of the host, as well as for the progression of tuberculosis [39,40]. Peptoids were therefore chosen for their potential ability to penetrate the bacterial cell wall similar to non-membrane peptides [13,22,23,28].

Second, several studies with peptide and peptoid analogs have shown that the more lipophilic the peptidomimetic (higher clogP), the more potent the peptoid due to its ability to either disrupt or translocate the bacterial cell wall. However, much higher lipophilicity results in greater toxicity. In fact, part of the safety problems and limited exploration of the full potential dose range associated with BDQ are related to its relatively high lipophilicity (measured logP of 7.25) [41]. For BDQ, this high lipophilicity likely accounts for its long terminal half-life (5–6 months), resulting in disproportional accumulation in tissues. Therefore, we hypothesized that β-peptoids with calculated clogP values of less than 7.0 would be lipophilic enough based on previously observed activity of peptidomimetics and yet not too lipophilic as to result in greater toxicity. Consequently, based on Ro5-compliant drugs (Lipinski's rule of 5), an upper limit of logP of 7.0 and topographical polar surface area (tPSA) of 140 Å2 were used as a general guide to design and select the peptoids in Table 1.

In this study, we use a phenotypic approach to test mycobacterial activity of extremely short synthetic alpha and beta-peptoids (1–3 residues) of varying lipophilicity. Modern phenotypic drug discovery (PDD) which combines modern tools and strategies based on realistic disease models has had some significant successes in the discovery of first-in-class drugs in recent years. The discovery of KAF156 for malaria, ivacaftor and lumicaftor for cystic fibrosis, crisaborole for atopic dermatitis, risdiplam and branaplam for spinal muscular atrophy, SEP-363856 for schizophrenia, among others [43,44], are examples of such recent successes of PDD.

Similarly, with regard to mycobacterial drug landscape, the target-based approach has not yielded a clinically useful anti-mycobacterial agent till date [45,46]. On the contrary, BDQ, one of only three anti-TB agents approved in more than 40 years, was discovered through PDD, starting with the initial identification of chemical agent by high throughput screening (HTS) using M. smegmatis as M. tb surrogate [46,47].

We hereby report a PDD-approach to the design, synthesis, and biological screening of ultra short alpha and beta-peptoids (1–3 residues) against M. tb and profiling of the most potent compounds against INH- and RMP-resistant M. tb strains, a range of Gram-negative and Gram-positive bacteria, mammalian cell cytotoxicity study, putative MOA specific to mycobacterial cell wall penetration, and combination study with commonly used anti-tubercular drugs.